RESUMO
OBJECTIVE: PTC-specific analysis identified novel fusions involving RET, BRAF, NTRK1, NTRK3, AGK and ALK genes in adults and pediatric PTCs. Although many novel fusions are PTC-specific events and, therefore, are ideal for diagnosis purposes, validation across additional and larger patient cohorts is essential for introducing these potential diagnostic or prognostic biomarkers into the clinical practice. As most of the BRAF, NTRK3 and ALK fusions were initially found in pediatric PTC or in more aggressive thyroid carcinomas, and there is a great disparity across population, in this study, we screened a large set of adult-sporadic PTC cases for the most prevalent kinase fusion lately described in the TCGA. DESIGN AND METHODS: The prevalence of the fusions was determined by RT-PCR in 71 classical PTC, 45 follicular variants of PTC (FVPTC), 19 follicular thyroid adenomas (FTAs) and 22 follicular thyroid carcinomas (FTCs). RESULTS: ETV6-NTRK3 was exclusively found in FVPTC, in both encapsulated and infiltrative variants, but was not found in FTAs and FTCs. STRN-ALK was found in both classical PTC and FVPTC. No AGK-BRAF fusion was identified in this series, endorsing that AGK-BRAF is a genetic event mainly associated with pediatric PTCs. CONCLUSIONS: The identification of kinase fusions in thyroid carcinomas helps to expand our knowledge about the landscape of oncogenic alterations in PTC. As ETV6-NTRK3 and STRN-ALK are recurrent and not identified in benign lesions, they can certainly help with diagnosis of thyroid nodules. Further analysis is needed to define if they can also be useful for prognosis and guiding therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Carcinoma Papilar/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkC/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adulto , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/genética , Proteínas de Ligação a Calmodulina/genética , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/genética , Estudos de Coortes , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas c-ets/genética , Receptores Proteína Tirosina Quinases/genética , Receptor trkC/genética , Proteínas Repressoras/genética , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Adulto Jovem , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
OsWRKY47 is a divergent rice transcription factor belonging to the group II of the WRKY family. A transcriptomic analysis of the drought response of transgenic rice plants expressing P SARK ::IPT, validated by qPCR, indicated that OsWRKY47 expression was induced under drought stress in P SARK ::IPT plants. A PCR-assisted site selection assay (SELEX) of recombinant OsWRKY47 protein showed that the preferred sequence bound in vitro is (G/T)TTGACT. Bioinformatics analyses identified a number of gene targets of OsWRKY47; among these two genes encode a Calmodulin binding protein and a Cys-rich secretory protein. Using Oswrk47 knockout mutants and transgenic rice overexpressing OsWRKY47 we show that the transcription of these putative targets were regulated by OsWRKY47. Phenotypic analysis carried out with transgenic rice plants showed that Oswrky47 mutants displayed higher sensitivity to drought and reduced yield, while plants overexpressing OsWRKY47 were more tolerant.
Assuntos
Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Secas , Técnicas de Inativação de Genes , Genes de Plantas , Dados de Sequência Molecular , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Estresse Fisiológico , TranscriptomaRESUMO
Age-related macular degeneration (AMD) causes visual impairment in the elderly. In non-neovascular AMD, studies involving human subjects have suggested potential involvement of aberrant lipid metabolism. However, there have been no reports on gene expression patterns in animal models of non-neovascular AMD with abnormal lipid metabolism such as apolipoprotein E knockout and human apolipoprotein E2 transgenic mice. Transcriptome analysis was performed using retinal pigment epithelium cells of apoE knockout and apolipoprotein E2 mice using microarray analysis. C57BL/6, Rxrb, Pparbp, Vldlr, and Edf1, which are primarily related to lipid metabolism, were upregulated, while Tgfbr1 and Pdgfb, which are related to pathologic angiogenesis in AMD, were downregulated in both types of mice. Apolipoprotein E knockout and apolipoprotein E2 mice showed characteristic gene expression patterns in the transcriptome analysis of primary retinal pigment epithelium cells. These results suggest that specific genes associated with lipid metabolism and angiogenesis are involved in the pathogenesis and progression of AMD.
Assuntos
Apolipoproteína E2/genética , Células Epiteliais/metabolismo , Epitélio Pigmentado da Retina/citologia , Transcriptoma , Idoso , Animais , Apolipoproteínas E/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Metabolismo dos Lipídeos , Linfocinas/genética , Linfocinas/metabolismo , Degeneração Macular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Análise em Microsséries , PPAR beta/genética , PPAR beta/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Regulação para CimaRESUMO
PURPOSE OF REVIEW: Aldosterone's functions and mechanisms of action are different depending on the tissue and the environmental condition. The mineralocorticoid receptor is present in tissues beyond epithelial cells, including the heart and vessels. Furthermore, aldosterone has direct adverse effects by both genomic and rapid/nongenomic actions not only through a nuclear receptor but also through caveolae-mediated intracellular events. Also, multiple environmental-genetic interactions play an important role in salt-sensitive hypertension (SSH) and aldosterone modulation. These findings have reshaped our vision of aldosterone's role in cardiovascular pathophysiology. This review describes new mediators of aldosterone's mechanisms of action: lysine-specific demethylase 1 (LSD1), caveolin 1 (cav-1) and striatin. RECENT FINDINGS: LSD1, an epigenetic regulator, is involved in the pathogenesis of SSH in both humans and rodents. In addition, cav-1, the main component of caveolae, plays a substantial role in mediating aldosterone pathways of SSH. The mineralocorticoid receptor interacts with cav-1 and is modulated by sodium intake. Finally, striatin, a scaffolding protein, mediates a novel interaction between signalling molecules and mineralocorticoid receptor's rapid effects in the cardiovascular system. SUMMARY: Substantial progress in aldosterone's functions and mechanisms of action should facilitate the study of cardiovascular diseases and the role of sodium intake in aldosterone-induced damage.
Assuntos
Aldosterona/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Doenças Cardiovasculares/enzimologia , Sistema Cardiovascular/enzimologia , Caveolinas/metabolismo , Histona Desmetilases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Epigenômica , Hemodinâmica , Humanos , Receptores de Mineralocorticoides/metabolismo , Cloreto de Sódio na Dieta/efeitos adversosRESUMO
Considerable effort has been invested in searching for less invasive methods of diagnosing endometriosis. Previous studies have indicated altered levels of the CALD1 gene (encoding the protein caldesmon) in endometriosis. The aims of our study were to investigate whether average CALD1 expression and caldesmon protein levels are differentially altered in the endometrium and endometriotic lesions and to evaluate the performance of the CALD1 gene and caldesmon protein as potential biomarkers for endometriosis. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from patients with endometriosis to evaluate CALD1 gene expression and caldesmon protein levels by real-time PCR and Western blot analysis, respectively. In addition, immunostaining for caldesmon to determine cellular localization was also performed. Endometrium from women without endometriosis was used as a control. Increased CALD1 expression and caldesmon levels were detected in the endometriotic lesions. The electrophoretic profile of caldesmon by Western blot analysis was clearly different between the control group (endometrium of women without endometriosis) and the group of women with endometriosis (eutopic endometrium and endometriotic lesions). Caldesmon expression as determined by immunostaining showed no variation among the cell types in endometriotic lesions and eutopic endometrium. Stromal cells marked positively in eutopic endometrium from control patients and in the endometriotic lesions. The presence of caldesmon in the endometrium of patients with and without endometriosis permitted diagnoses with 95% sensitivity (specificity 100%) and 100% sensitivity (specificity 100%) for the disease and for minimal to mild endometriosis in the proliferative phase of the menstrual cycle, respectively. In the secretory phase, minimal to mild endometriosis was detected with 90% sensitivity and 93.3% specificity. Caldesmon is a possible predictor of endometrial dysregulation in patients with endometriosis. A potential limitation of our study is the fact that other endometrial diseases were not excluded, and therefore prospective studies are needed to confirm the potential of caldesmon as a biomarker exclusively for endometriosis.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Endometriose/diagnóstico , Endométrio/metabolismo , Doenças Ovarianas/diagnóstico , Doenças Peritoneais/diagnóstico , Adulto , Biomarcadores/metabolismo , Proteínas de Ligação a Calmodulina/genética , Estudos de Casos e Controles , Endometriose/genética , Endometriose/metabolismo , Endométrio/patologia , Feminino , Humanos , Doenças Ovarianas/genética , Doenças Ovarianas/metabolismo , Doenças Peritoneais/genética , Doenças Peritoneais/metabolismo , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
The endothelial differentiation factor-1 (EDF-1) is a calmodulin binding protein that regulates calmodulin-dependent enzymes. In endothelial cells, this factor can form a protein complex with calmodulin. We analyzed the relationship between this factor and the members of calmodulin/calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway during adipogenesis of 3T3-F442A cells. We found that the expression of edf1 is upregulated during early adipogenesis, whereas that of calcineurin gene is lowered, suggesting that this pathway should be downregulated to allow for adipogenesis to occur. We also found that EDF-1 associates with calmodulin and calcineurin, most likely inactivating calcineurin. Our results showed that EDF-1 inactivates the calmodulin/calcineurin/NFAT pathway via sequestration of calmodulin, during early adipogenesis, and we propose a mechanism that negatively regulates the activation of calcineurin through a complex formation between EDF-1 and calmodulin. This finding raises the possibility that modulating this pathway might offer some alternatives to regulate adipose biology.
Assuntos
Adipogenia/fisiologia , Calcineurina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Adipogenia/genética , Animais , Calcineurina/genética , Inibidores de Calcineurina , Calmodulina/genética , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Regulação para Baixo , Camundongos , Fatores de Transcrição NFATC/genética , Transdução de SinaisRESUMO
Calmodulin (CaM) is the primary sensor for calcium in the cell. It modulates various functions by activating CaM-binding proteins (CaMBPs). This study examined the calcium/CaM-dependent system in the ancient eukaryote Giardia intestinalis. A specific antibody against the parasite's CaM was developed; this protein's expression and location during different stages of the parasite's life cycle were analyzed. The results showed that it is a housekeeping protein which is possibly involved in the parasite's motility. No CaMBP has been identified in G. intestinalis to date. Pull-down assays were used for isolating proteins which specifically bind to CaM in a calcium-dependent way. Three of them were identified through mass spectrometry; they were GASP180, α-tubulin, and pyruvate phosphate dikinase (PPDK).The first two are cytoskeleton proteins, and the last one is an essential enzyme for glycolysis. The presence of binding sites was analyzed through bioinformatics in each protein sequence. This is the first report of a CaMBP in this organism; it is considered to be a very interesting differentiation model, indicating that CaM is involved at least in two vital processes: G. intestinalis motility and energetic metabolism.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Giardia lamblia/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Técnicas de Cultura de Células , Diferenciação Celular , Movimento Celular , Biologia Computacional , Giardia lamblia/metabolismo , Filogenia , Processamento de Proteína Pós-Traducional , Piruvato Ortofosfato Diquinase/metabolismo , Tubulina (Proteína)RESUMO
The Ca(2+)/calmodulin complex interacts with and regulates various enzymes and target proteins known as calmodulin-binding proteins (CaMBPs). This group of proteins includes molecular motors such as myosins. In this study, we show that non-muscle myosin-IIB is overexpressed in the brains of diabetic rats. We isolated CaMBPs from the brains of non-diabetic rats and rats with streptozotocin-induced diabetes and purified them by immobilized-calmodulin affinity chromatography. The proteins were eluted with EGTA and urea, separated by SDS-PAGE, digested and submitted to peptide mass fingerprinting analysis. Thirteen intense bands were found in both types of brains, two were found exclusively in non-diabetic brains and four were found exclusively in diabetic brains. A large fraction of the eluted proteins contained putative IQ motifs or calmodulin-binding sites. The results of the myosin-IIB affinity chromatography elution, western blot and RT-PCR analyses suggest that myosin-IIB protein and mRNA are expressed at high levels in diabetic brains. This is the first study that has demonstrated differential expression of CaMBPs in diabetic and non-diabetic brain tissue through a comparative proteomic analysis, and it opens up a new approach to studying the relationship between the expression of myosins in the brain, hyperglycemia and intracellular calcium regulation.
Assuntos
Química Encefálica/fisiologia , Diabetes Mellitus Experimental/metabolismo , Miosina não Muscular Tipo IIB/biossíntese , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Ligação a Calmodulina/metabolismo , Cromatografia de Afinidade , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , RNA/biossíntese , RNA/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/químicaRESUMO
Calmodulin is a Ca(+2)-binding protein important in a variety of cell functions. The Ca(+2)/calmodulin complex interacts with and regulates various enzymes and target proteins, known as calmodulin-binding proteins (CaMBPs). In this study, we revealed a comparative identification of the CaMBPs composition in the worker honeybee (Apis mellifera) brain, considering two different honeybee behaviors in the colony. To this end, the CaMBPs of forager and nurse workers were purified by affinity chromatography, separated in 1D gel, digested and submitted to peptide mass fingerprinting (PMF). In the PMF analysis, 15 different proteins, considered behavior-specific proteins, were identified, one of them exclusively in forager workers and 10 in nurses. All the proteins were classified in terms of their function and cell localization, revealing a greater expression of metabolism-related CaMBPs in both worker subcastes. Protein sequences were then analyzed for the presence of the calmodulin-binding sites. Therefore, the honeybee brain CaMBPs profiles presented differences between worker subcastes. This is the first identification of calmodulin-binding proteins in the brain of A. mellifera upon nursing and foraging behaviors in the colony and this diversity of target proteins for Ca(+2)/CaM may be involved in terms of the function of these proteins in the nervous system.
Assuntos
Abelhas/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Motivos de Aminoácidos , Animais , Encéfalo/citologia , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/classificação , Vesículas Citoplasmáticas/metabolismo , Citoesqueleto/metabolismoRESUMO
We have previously shown that the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet-intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non-erythroid spectrin). Here we demonstrate that Pet has affinity for alpha-fodrin (formally named alphaII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST-fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain. Site-directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet-treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST-fodrin. This is the first report showing cleavage of alpha-fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.