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1.
DNA Cell Biol ; 38(10): 1100-1111, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418589

RESUMO

Members of the Sox gene family play crucial roles during reproduction and development, but their genome-wide identification has not yet been performed in large yellow croaker, Larimichthys crocea. In this study, a total of 26 members of the Sox gene family were identified from the genome of large yellow croaker and classified into seven subgroups based on the conserved HMG-box domain they contain. Among the identified Sox gene family members, eight belonged to the SoxB subgroup (five in B1 and three in B2), four belonged to the SoxC subgroup, four belonged to the SoxD subgroup, six belonged to the SoxE subgroup, three belonged to the SoxF subgroup, and one belonged to the SoxK subgroup. During evolution, members of the SoxE subgroup (Sox8, Sox9, Sox10), Sox1, Sox4, Sox6, and Sox11 evolved into two copies, which may be a result of teleost-specific whole-genome duplication. Sox genes were distributed unevenly across 15 chromosomes. The number of introns in large yellow croaker Sox genes varied from 0 to 14. Results of the expression profile during embryogenesis revealed that most of the members of the Sox gene family had lower expression, except several Sox genes, and expression patterns also differed among each Sox gene group and duplicated gene. This study systematically characterized and analyzed the Sox gene family in large yellow croaker and provided new insights into its function during embryogenesis.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Perciformes/genética , Filogenia , Fatores de Transcrição SOXB1/genética , Sequência de Aminoácidos , Animais , Evolução Biológica , Mapeamento Cromossômico , Biologia Computacional , Embrião não Mamífero , Desenvolvimento Embrionário , Éxons , Proteínas de Peixes/classificação , Duplicação Gênica , Íntrons , Família Multigênica , Perciformes/classificação , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Fatores de Transcrição SOXB1/classificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
2.
BMC Bioinformatics ; 20(1): 434, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438847

RESUMO

BACKGROUND: The epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells. Elevated expression levels of EGFR are associated with prognosis and clinical outcomes of patients in a variety of tumor types. There are at least four splice variants of the mRNA encoding four protein isoforms of EGFR in humans, named I through IV. EGFR isoform I is the full-length protein, whereas isoforms II-IV are shorter protein isoforms. Nevertheless, all EGFR isoforms bind the epidermal growth factor (EGF). Although EGFR is an essential target of long-established and successful tumor therapeutics, the exact function and biomarker potential of alternative EGFR isoforms II-IV are unclear, motivating more in-depth analyses. Hence, we analyzed transcriptome data from glioblastoma cell line SF767 to predict target genes regulated by EGFR isoforms II-IV, but not by EGFR isoform I nor other receptors such as HER2, HER3, or HER4. RESULTS: We analyzed the differential expression of potential target genes in a glioblastoma cell line in two nested RNAi experimental conditions and one negative control, contrasting expression with EGF stimulation against expression without EGF stimulation. In one RNAi experiment, we selectively knocked down EGFR splice variant I, while in the other we knocked down all four EGFR splice variants, so the associated effects of EGFR II-IV knock-down can only be inferred indirectly. For this type of nested experimental design, we developed a two-step bioinformatics approach based on the Bayesian Information Criterion for predicting putative target genes of EGFR isoforms II-IV. Finally, we experimentally validated a set of six putative target genes, and we found that qPCR validations confirmed the predictions in all cases. CONCLUSIONS: By performing RNAi experiments for three poorly investigated EGFR isoforms, we were able to successfully predict 1140 putative target genes specifically regulated by EGFR isoforms II-IV using the developed Bayesian Gene Selection Criterion (BGSC) approach. This approach is easily utilizable for the analysis of data of other nested experimental designs, and we provide an implementation in R that is easily adaptable to similar data or experimental designs together with all raw datasets used in this study in the BGSC repository, https://github.com/GrosseLab/BGSC .


Assuntos
Processamento Alternativo/genética , Biologia Computacional/métodos , Receptores ErbB/genética , Glioblastoma/genética , Teorema de Bayes , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Probabilidade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
3.
Arch Virol ; 164(11): 2793-2797, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31440811

RESUMO

The DC-SIGN glycoprotein is responsible for the initial adhesion of dengue virus (DENV) to immune cells by the carbohydrate recognition domain (CRD). There are thirteen soluble and membrane-bound DC-SIGN isoforms, but the role of soluble isoforms in the DENV internalization process is not known. Five isoforms with an altered or absent CRD were identified, and three different soluble isoforms were used to confirm the interactions with mannose residues. The results show the loss of binding ability of one soluble isoform and binding ability of two of them. All of them will be used to verify their role in the DENV internalization process.


Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Vírus da Dengue/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Manose/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Ligação Viral , Internalização do Vírus , Sequência de Aminoácidos , Sequência de Bases , Dengue/virologia , Vírus da Dengue/genética , Ligantes , Ligação Proteica/genética , Isoformas de Proteínas/genética
4.
Nat Neurosci ; 22(10): 1709-1717, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451803

RESUMO

Nervous system function relies on complex assemblies of distinct neuronal cell types that have unique anatomical and functional properties instructed by molecular programs. Alternative splicing is a key mechanism for the expansion of molecular repertoires, and protein splice isoforms shape neuronal cell surface recognition and function. However, the logic of how alternative splicing programs are arrayed across neuronal cells types is poorly understood. We systematically mapped ribosome-associated transcript isoforms in genetically defined neuron types of the mouse forebrain. Our dataset provides an extensive resource of transcript diversity across major neuron classes. We find that neuronal transcript isoform profiles reliably distinguish even closely related classes of pyramidal cells and inhibitory interneurons in the mouse hippocampus and neocortex. These highly specific alternative splicing programs selectively control synaptic proteins and intrinsic neuronal properties. Thus, transcript diversification via alternative splicing is a central mechanism for the functional specification of neuronal cell types and circuits.


Assuntos
Processamento Alternativo/genética , Neurônios/fisiologia , Ribossomos/genética , Transcrição Genética/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neurônios/classificação , Terminações Pré-Sinápticas/metabolismo , Prosencéfalo/citologia , Isoformas de Proteínas/genética , Células Piramidais/fisiologia
5.
Life Sci ; 234: 116768, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445027

RESUMO

In prostate cancer development, the androgen receptor (AR) signaling plays a crucial role during both formation of early prostate lesions and progression to the lethal, incurable castration resistant stage. Accordingly, numerous approaches have been developed to inhibit AR activity including androgen deprivation therapy, application of the AR antagonists as well as the use of taxanes. However, these treatments, although effective initially, resistance inevitably occur for most of the patients within several years and limiting the therapeutic efficacy. Of note, alterations and reactivation of the AR signaling pathway have been demonstrated as the major reasons for the observed resistance. Accumulating evidences have suggested that synthesis of AR splicing variants, in particular, the constitutively active AR-V7, is one of the most important mechanisms that contribute to the abnormal AR signaling. In addition, clinical data also highlight the potential of using AR-V7 as a predictive biomarker and a therapeutic target in metastatic castration resistant prostate cancer (mCRPC). In this review, we summarize the recent findings concerning the specific role of AR-V7 in CRPC progression, drug resistance and its potential value in clinical assessment.


Assuntos
Antagonistas de Receptores de Andrógenos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Taxoides/uso terapêutico , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Antineoplásicos/farmacologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxoides/farmacologia
6.
Comput Biol Chem ; 82: 37-43, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31255973

RESUMO

Tubulin protein is the fundamental unit of microtubules, and comprises of α and ß subunits arranged in an alternate manner forming protofilaments. These longitudinal protofilaments are made up of intra- (α-ß) and inter-dimer (ß-α) interactions. Literature review confirms that GTP hydrolysis results in considerable structural rearrangement within GTP binding site of ß-α dimer interface after the release of γ phosphate. In addition to this, the intra-dimer interface exhibits structural rigidity which needs further investigation. In this study, we explored the reasons for the flexibility and the rigidity of the ß-α dimer and the α-ß dimer respectively through molecular simulation and Anisotropic Normal Mode based analysis. As per the sequence alignment report, two glycine residues (Gly96 and Gly98) were observed in the T3 loop of the ß subunit which get substituted by Asp98 and Ala100 in the T3 loop of the α subunit. The higher mobility of glycine residues contributes to the flexibility of the T3 loop of inter-dimer when they come in direct contact with the GTPase Activating Protein (GAP) domain of the subunit. This was confirmed through RMSD, RMSF and Radius of Gyration based studies. Conversely, the intra-dimer exhibited a lower mobility in the absence of glycine residues. As per ANM based analysis, positive domain correlations were observed between T3 loop and GAP domain of intra- and inter- dimeric contact regions. However, these correlation motions were higher in the intra-dimer as compared to the inter-dimer interface. Thus on the basis of our findings, we hypothesize that the higher flexibility of T3 loop and the GAP domain of the inter-dimer is required for structural rearrangement and protofilament stability during hydrolysis. Furthermore, the slightly rigid nature of the T3 loop and the GAP domain of the intra-dimer assists in enhancing the monomer-monomer interaction through the higher positive domain correlation.


Assuntos
Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Anisotropia , Sítios de Ligação , Bovinos , Glicina/química , Simulação de Dinâmica Molecular , Mutação , Maleabilidade , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Alinhamento de Sequência , Tubulina (Proteína)/genética
7.
Gene ; 714: 144004, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351124

RESUMO

Calreticulin (CRT) is calcium binding protein of endoplasmic reticulum (ER) which performs plethora of functions besides it's role as molecular chaperone. Among the three different isoforms of this protein, CRT3 is most closely related to primitive CRT gene of higher plants. Based on their distinct structural and functional organisation, the plant CRTs have been known to contain three different domains: N, P and the C domain. The domain organisation and various biochemical characterstics of plant and animal CRTs are common with the exception of some differences. In plant calreticulin, the important N-glycosylation site(s) are replaced by the glycan chain(s) and several consensus sequences for in vitro phosphorylation by protein kinase CK2 (casein kinase-2), are also present unlike the animal calreticulin. Biotic and abiotic stresses play a significant role in bringing down the crop production. The role of various phytohormones in defense against fungal pathogens is well documented. CRT3 has been reported to play important role in protecting the plants against fungal and bacterial pathogens and in maintaining plant innate immunity. There is remarkable crosstalk between CRT mediated signalling and biotic, abiotic stress, and phytohormone mediated signalling pathways The role of CRT mediated pathway in mitigating biotic and abiotic stress can be further explored in plants so as to strategically modify it for development of stress tolerant plants.


Assuntos
Proteínas de Arabidopsis/genética , Calreticulina/genética , Transdução de Sinais/genética , Estresse Fisiológico/genética , Animais , Regulação da Expressão Gênica de Plantas/genética , Imunidade Vegetal/genética , Isoformas de Proteínas/genética
8.
Cell Mol Life Sci ; 76(20): 3969-3985, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31218451

RESUMO

Cardiovascular diseases represent one of the most important health problems of developed countries. One of the main actors involved in the onset and development of cardiovascular diseases is the increased production of reactive oxygen species that, through lipid peroxidation, protein oxidation and DNA damage, induce oxidative stress and cell death. Basic and clinical research are ongoing to better understand the endogenous antioxidant mechanisms that counteract oxidative stress, which may allow to identify a possible therapeutic targeting/application in the field of stress-dependent cardiovascular pathologies. In this context, increasing attention is paid to the glutathione/glutathione-peroxidase and to the thioredoxin/thioredoxin-reductase systems, among the most potent endogenous antioxidative systems. These key enzymes, belonging to the selenoprotein family, have a well-established function in the regulation of the oxidative cell balance. The aim of the present review was to highlight the role of selenoproteins in cardiovascular diseases, introducing the emerging cardioprotective role of endoplasmic reticulum-resident members and in particular one of them, namely selenoprotein T or SELENOT. Accumulating evidence indicates that the dysfunction of different selenoproteins is involved in the susceptibility to oxidative stress and its associated cardiovascular alterations, such as congestive heart failure, coronary diseases, impaired cardiac structure and function. Some of them are under investigation as useful pathological biomarkers. In addition, SELENOT exhibited intriguing cardioprotective effects by reducing the cardiac ischemic damage, in terms of infarct size and performance. In conclusion, selenoproteins could represent valuable targets to treat and diagnose cardiovascular diseases secondary to oxidative stress, opening a new avenue in the field of related therapeutic strategies.


Assuntos
Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peptídeos/uso terapêutico , Selenocisteína/metabolismo , Selenoproteínas/genética , Animais , Antioxidantes/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/terapia , Regulação da Expressão Gênica , Glutationa Peroxidase/metabolismo , Humanos , Terapia de Alvo Molecular/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Selenoproteínas/agonistas , Selenoproteínas/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo
9.
Cell Mol Life Sci ; 76(20): 4023-4042, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31236625

RESUMO

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.


Assuntos
Mitocôndrias/enzimologia , Neoplasias/enzimologia , Doenças Neurodegenerativas/enzimologia , Subunidades Proteicas/química , Proteínas/genética , Succinato Desidrogenase/química , Animais , Ciclo do Ácido Cítrico/genética , Coenzimas/química , Coenzimas/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fosforilação Oxidativa , Plantas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
10.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
11.
Nat Commun ; 10(1): 2767, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235694

RESUMO

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications.


Assuntos
Metabolismo Energético/fisiologia , Fadiga/fisiopatologia , Cinurenina/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Adaptação Fisiológica , Animais , Aspartato Aminotransferases/metabolismo , Ácido Aspártico/metabolismo , Carbidopa/farmacologia , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Glicólise/fisiologia , Malatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Animais , Músculo Esquelético/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Condicionamento Físico Animal/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transaminases/antagonistas & inibidores , Transaminases/metabolismo
12.
Biochemistry (Mosc) ; 84(4): 329-345, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228925

RESUMO

The review describes functional and structural features of different isoforms of prolactin receptor, mechanisms of signaling pathway activation, and molecular messengers involved in the transmission and termination of signal from the prolactin receptor isoforms. Changes in the ratio between prolactin receptor isoforms, key mediators of prolactin signal transduction and termination in various organs and tissues, are analyzed. Special attention is given to the role of molecular mediators and the ratio between the isoforms in normal physiological functions and pathologies. Approaches for therapeutic correction of prolactin signaling impairments are discussed.


Assuntos
Prolactina/metabolismo , Receptores da Prolactina/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores da Prolactina/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo
13.
Folia Histochem Cytobiol ; 57(2): 84-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31198984

RESUMO

INTRODUCTION: WNT5A (Wnt family member 5A) belongs to the WNT family of secreted signaling glycoproteins that play essential role in developmental, physiological and pathological processes. WNT5A was shown to take part in carcinogenesis process playing both oncogenic and suppressor functions in various types of human malignancies. This study aimed to assess the expression of the WNT5A gene at the mRNA and protein levels in the specimens derived from endometrial cancer (EC) or unchanged control endometrium. The associations between the WNT5A expression levels and clinicopathological characteristics and survival of EC patients were evaluated. MATERIALS AND METHODS: Total RNA was isolated in order to assess the relative amounts of WNT5A mRNA by quantitative polymerase chain reaction (QPCR) in samples of unchanged endometrial control (n = 8) and tumor samples of EC patients (n = 28). Immunohistochemistry (IHC) was used to determine the presence of WNT5A protein in the sections of formalin-fixed, paraffin-embedded tissue specimens derived from unchanged endome-trial controls (n = 6) and EC tumors (n = 19). Significance of differences in WNT5A expression levels between the studied groups of EC patients and correlations between the WNT5A and demographic data, pathological features, hematological parameters and overall survival of the patients were evaluated by statistical analysis. RESULTS: The level of WNT5A mRNA was decreased in EC in comparison to unchanged endometrium. WNT5A expression was associated with primary tumor invasion status exhibiting reduced level of transcripts in EC that involved organs beyond the uterus when compared to the uterus-confined cancers. WNT5A immunoreactivity was visualized in the cytoplasm and nuclei of EC cells as well as in the luminal and glandular epithelial cells of unchanged endometrium. WNT5A mRNA expression levels correlated negatively with cytoplasmic, and positively with nuclear immunoreactivity of the WNT5A protein in the EC cells. In addition, the relationships between blood leucocyte count (in particular granulocytes and lymphocytes) of patients with EC and their WNT5A mRNA and protein expression levels were established. A positive correlation between the nuclear immunoexpression of WNT5A protein in the cancer cells in cell nuclei and mean platelet volume in blood was also found. CONCLUSIONS: The results of the first study of WNT5A expression at the transcript and protein levels indicate that it could be considered as a potential marker of molecular changes that take place during EC development.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , RNA Mensageiro/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/mortalidade , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Proteína Wnt-5a/imunologia
14.
J Toxicol Sci ; 44(5): 327-333, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068538

RESUMO

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, and metal-binding protein that protects cells from the cytotoxic effects of heavy metals and reactive oxygen species. Previously, we found that transcriptional induction of endothelial MT-1A was mediated by not only the metal-regulatory transcription factor 1 (MTF-1)-metal responsive element (MRE) pathway but also the nuclear factor-erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile responsive element (ARE) pathway, whereas that of MT-2A was mediated only by the MTF-1-MRE pathway, using the organopnictogen compounds tris(pentafluorophenyl)stibane, tris(pentafluorophenyl)arsane, and tris(pentafluorophenyl)phosphane as molecular probes in vascular endothelial cells. In the present study, we investigated the binding sites of MTF-1 and Nrf2 in the promoter regions of MTs in cultured bovine aortic endothelial cells treated with these organopnictogen compounds. We propose potential mechanisms underlying transcriptional induction of endothelial MT isoforms. Specifically, both MRE activation by MTF-1 and that of ARE in the promoter region of the MT-2A gene by Nrf2 are involved in transcriptional induction of MT-1A, whereas only MRE activation by MTF-1 or other transcriptional factor(s) is required for transcriptional induction of MT-2A in vascular endothelial cells.


Assuntos
Células Endoteliais/efeitos dos fármacos , Metalotioneína/genética , Fosfinas/toxicidade , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Isoformas de Proteínas/genética , Fatores de Transcrição/genética , Transcrição Genética
15.
Neuron ; 103(1): 66-79.e12, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31104951

RESUMO

The precision and reliability of synaptic information transfer depend on the molecular organization of voltage-gated calcium channels (VGCCs) within the presynaptic membrane. Alternative splicing of exon 47 affects the C-terminal structure of VGCCs and their affinity to intracellular partners and synaptic vesicles (SVs). We show that hippocampal synapses expressing VGCCs either with exon 47 (CaV2.1+47) or without (CaV2.1Δ47) differ in release probability and short-term plasticity. Tracking single channels revealed transient visits (∼100 ms) of presynaptic VGCCs in nanodomains (∼80 nm) that were controlled by neuronal network activity. Surprisingly, despite harboring prominent binding sites to scaffold proteins, CaV2.1+47 persistently displayed higher mobility within nanodomains. Synaptic accumulation of CaV2.1 was accomplished by optogenetic clustering, but only CaV2.1+47 increased transmitter release and enhanced synaptic short-term depression. We propose that exon 47-related alternative splicing of CaV2.1 channels controls synapse-specific release properties at the level of channel mobility-dependent coupling between VGCCs and SVs.


Assuntos
Canais de Cálcio/genética , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/efeitos da radiação , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Células HEK293 , Humanos , Luz , Neurotransmissores/metabolismo , Optogenética , Gravidez , Isoformas de Proteínas/genética , Ratos , Vesículas Sinápticas/fisiologia
16.
Gene ; 709: 17-24, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31102716

RESUMO

Angiopoietin-like protein 6, which is encoded by ANGPTL6 gene (also known as angiopoietin growth factor, AGF), has been extensively characterized with regard to its proposed functions as angiogenesis and energy metabolism. The present results showed the occurrence of alternative splicing by intron retention (IR) event in the bovine ANGPTL6 gene (bANGPTL6). By means of RT-PCR, TA clone and sequencing, we have shown that the bANGPTL6 gene has a splice variant generated by the retention of its partial intron 3. The computational analysis of the bANGPTL6 genomic sequence showed that its intron 3 has a high percentage of GC (62.31%) and a length of 199 nt, characteristics that have been associated with an IR event. The IR event does not interfere with the coding region as the bANGPTL6 prepropeptide is entirely coded in the third exon. Additionally, both the intronless (namely, bANGPTL6α) and intron-retaining (namely, bANGPTL6ß) ANGPTL6 transcripts are constitutively co-expressed in the bovine liver. Further, the relative expression level of different variants in liver was tested by both semi-RT-PCR and RT-qPCR methods. The results suggested bANGPTL6ß are significantly higher than bANGPTL6α. Overall, our findings will be helpful for studies on the molecular mechanism of IR events and the functions of ANGPTL6 gene. Specially, bANGPTL6ß gene probably contributes to a new target for treatment of obesity and obesity-related diseases.


Assuntos
Processamento Alternativo/genética , Proteínas Semelhantes a Angiopoietina/genética , Bovinos/genética , Íntrons/genética , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Expressão Gênica , Fígado/metabolismo , Isoformas de Proteínas/genética
17.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091671

RESUMO

The human Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane transport protein that plays an important role in pH regulation in mammalian cells. Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, protons accumulate within the cytosol. Extracellular signal-regulated kinase (ERK)-mediated regulation of NHE1 is important in several human pathologies including in the myocardium in heart disease, as well as in breast cancer as a trigger for growth and metastasis. NHE1 has a N-terminal, a 500 amino acid membrane domain, and a C-terminal 315 amino acid cytosolic domain. The C-terminal domain regulates the membrane domain and its effects on transport are modified by protein binding and phosphorylation. Here, we discuss the physiological regulation of NHE1 by ERK, with an emphasis on the critical effects on structure and function. ERK binds directly to the cytosolic domain at specific binding domains. ERK also phosphorylates NHE1 directly at multiple sites, which enhance NHE1 activity with subsequent downstream physiological effects. The NHE1 cytosolic regulatory tail possesses both ordered and disordered regions, and the disordered regions are stabilized by ERK-mediated phosphorylation at a phosphorylation motif. Overall, ERK pathway mediated phosphorylation modulates the NHE1 tail, and affects the activity, structure, and function of this membrane protein.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Humanos , Fosforilação , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/química , Trocador 1 de Sódio-Hidrogênio/genética
18.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071995

RESUMO

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aß peptide in amyloid plaques in Alzheimer's disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer's disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE-Αß complex. Our in vitro biophysical results prove that apoE peptide-analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE-Αß complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Αß; taking an important step forward in the field of Alzheimer's anti-aggregation drug development.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Amiloidose/genética , Apolipoproteínas E/química , Doença de Alzheimer/patologia , Amiloide/química , Amiloide/genética , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/ultraestrutura , Amiloidose/patologia , Apolipoproteínas E/ultraestrutura , Sítios de Ligação , Colesterol/química , Colesterol/genética , Humanos , Placa Amiloide/genética , Placa Amiloide/patologia , Placa Amiloide/ultraestrutura , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestrutura
19.
Anal Chim Acta ; 1067: 129-136, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31047144

RESUMO

BCR/ABLp210 fusion gene, the characteristic biomarker of chronic myelogenous leukemia (CML), contains two different transcription isoforms, e13a2 and e14a2, which lead to differences in the pathological features and response to targeted drug. At present, there is short of simple and fast technology to distinguish these two transcript isoforms. In this paper, RNA fusion-triggered rolling circle amplification (RF-RCA) strategy was developed to distinguish e13a2 and e14a2 transcripts directly from RNA extraction in one step. The simultaneous binding of dumbbell template and corresponding primer with target fused RNA can induce their proximal hybridization and trigger the RCA to produce lots of tandem repeat G-quadruplexes sequences for real time fluorescence readout with the interaction of Thioflavin T and G-quadruplex. The proposed strategy can detect as low as 0.1 aM target and discriminate e13a2 (0.01%) and e14a2 (0.1%) transcript isoforms directly from complex genomic RNA extraction, proving high sensitivity and specificity. Furthermore, the RF-RNA was successfully applied to analyze BCR/ABLp210 isoforms from clinical samples for accurately molecular subtyping and monitoring the response of imatinib treatment. The developed RF-RCA strategy presented an ultrasensitive, accurate and pragmatic toolbox to simple and rapid discriminate BCR/ABLp210 fusion isoforms for promoting clinical research and personalized treatment of CML.


Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Técnicas de Amplificação de Ácido Nucleico , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Isoformas de Proteínas/genética , Ativação Transcricional/genética
20.
Nat Cell Biol ; 21(5): 640-650, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011167

RESUMO

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Fator de Processamento U2AF/genética , Processamento Alternativo/genética , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata/genética , Inflamação/genética , Inflamação/patologia , Leucemia Mieloide Aguda/patologia , Masculino , Mutação/genética , Síndromes Mielodisplásicas/patologia , Isoformas de Proteínas/genética , Transdução de Sinais , Spliceossomos/genética
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