RESUMO
Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.
Assuntos
Cardiomiopatia Chagásica , Conexina 43 , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Conexina 43/metabolismo , Conexina 43/genética , Animais , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Miócitos Cardíacos/patologia , Inflamação/metabolismo , Fosforilação , Masculino , Doença Crônica , Trypanosoma cruzi , Modelos Animais de Doenças , Linhagem Celular , Citocinas/metabolismo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/parasitologia , Arritmias Cardíacas/imunologia , FemininoRESUMO
The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.
Assuntos
Apoptose , Proteínas Quinases Dependentes de AMP Cíclico , Células Epiteliais , Ciclo Estral , Transdução de Sinais , Animais , Feminino , Células Epiteliais/metabolismo , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo Estral/fisiologia , Ciclo Estral/metabolismo , Oviductos/metabolismo , Oviductos/citologia , Tubas Uterinas/metabolismo , Tubas Uterinas/citologia , FosforilaçãoRESUMO
In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Quebras de DNA de Cadeia Dupla , NF-kappa B , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Camundongos , Fosforilação , NF-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Camundongos Knockout , Etoposídeo/farmacologia , Motivos de AminoácidosRESUMO
The tumor cells reprogram their metabolism to cover their high bioenergetic demands for maintaining uncontrolled growth. This response can be mediated by cytokines such as IL-2, which binds to its receptor and activates the JAK/STAT pathway. Some reports show a correlation between the JAK/STAT pathway and cellular metabolism, since the constitutive activation of STAT proteins promotes glycolysis through the transcriptional activation of genes related to energetic metabolism. However, the role of STAT proteins in the metabolic switch induced by cytokines in cervical cancer remains poorly understood. In this study, we analyzed the effect of IL-2 on the metabolic switch and the role of STAT5 in this response. Our results show that IL-2 induces cervical cancer cell proliferation and the tyrosine phosphorylation of STAT5. Also, it induces an increase in lactate secretion and the ratio of NAD+/NADH, which suggest a metabolic reprogramming of their metabolism. When STAT5 was silenced, the lactate secretion and the NAD+/NADH ratio decreased. Also, the expression of HIF1α and GLUT1 decreased. These results indicate that STAT5 regulates IL-2-induced cell proliferation and the metabolic shift to aerobic glycolysis by regulating genes related to energy metabolism. Our results suggest that STAT proteins modulate the metabolic switch in cervical cancer cells to attend to their high demand of energy required for cell growth and proliferation.
Assuntos
Proliferação de Células , Interleucina-2 , Fator de Transcrição STAT5 , Neoplasias do Colo do Útero , Humanos , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/genética , Feminino , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Glicólise/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , NAD/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transdução de Sinais/efeitos dos fármacos , Ácido Láctico/metabolismoRESUMO
Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.
Assuntos
AMP Cíclico , Capacitação Espermática , Espermatozoides , Proteína com Valosina , Animais , Masculino , Capacitação Espermática/efeitos dos fármacos , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Espermatozoides/metabolismo , Camundongos , AMP Cíclico/metabolismo , Fosforilação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.
Assuntos
Capacitação Espermática , Espermatozoides , Animais , Capacitação Espermática/efeitos dos fármacos , Masculino , Cavalos/fisiologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Cálcio/metabolismo , Zona Pelúcida/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , FosforilaçãoRESUMO
INTRODUCTION: AMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. METHODS: We studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. RESULTS: Cellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. CONCLUSION: Our data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.
Assuntos
Proteínas Quinases Ativadas por AMP , Adenoma , Adenoma Hipofisário Secretor de Hormônio do Crescimento , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Masculino , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Feminino , Pessoa de Meia-Idade , Adulto , Adenoma/genética , Adenoma/patologia , Adenoma/metabolismo , Adenoma/enzimologia , Acromegalia/genética , Acromegalia/patologia , Acromegalia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Idoso , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/enzimologia , Fosforilação , Hipófise/metabolismo , Hipófise/patologia , Regulação Neoplásica da Expressão GênicaRESUMO
Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated in vivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.
Assuntos
Envelhecimento , Proteínas Quinases Dependentes de AMP Cíclico , Dieta Cetogênica , Potenciação de Longa Duração , Memória , Proteoma , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Envelhecimento/fisiologia , Envelhecimento/metabolismo , Dieta Cetogênica/métodos , Proteoma/metabolismo , Camundongos , Masculino , Memória/fisiologia , Potenciação de Longa Duração/fisiologia , Camundongos Endogâmicos C57BL , Hipocampo/metabolismo , Sinapses/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Plasticidade Neuronal/fisiologia , FosforilaçãoRESUMO
The aim of this study was to develop a star fruit extract (SFE) and incorporate it into aerogels based on native and phosphorylated potato starches. The phosphorylation of starch enhances its properties by incorporating phosphate groups that increase the spaces between starch molecules, resulting in a more resilient, intact aerogel with enhanced water absorption. The bioactive aerogels based on potato starch and 10, 15, and 20 % (w/w) of SFE were characterized by their morphological and thermogravimetric properties, infrared spectra, water absorption capacity, loading capacity, and antioxidant activity. Epicatechin was the major compound present in SFE. The thermal stability of SFE increased when incorporated into phosphorylated starch aerogels at a concentration of 20 %. The water absorption capacity was higher in phosphorylated starch aerogels (reaching 1577 %) than in their native counterparts (reaching 1100 %). Native starch aerogels with 15 and 20 % SFE exhibited higher antioxidant activity against hydroxyl free radicals compared to phosphorylated starch aerogels, achieving 79.9 % and 86.4 % inhibition for the hydroxyl and nitric oxide radicals, respectively. The ideal choice of freeze-dried aerogel depends on the desired effect, either to act as an antioxidant agent by releasing bioactive compounds from SFE or as a water-absorbent agent in food products.
Assuntos
Antioxidantes , Frutas , Géis , Extratos Vegetais , Solanum tuberosum , Amido , Solanum tuberosum/química , Géis/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Amido/química , Fosforilação , Antioxidantes/química , Antioxidantes/farmacologia , Frutas/química , Averrhoa/química , Água/químicaRESUMO
LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid ß-arrestin-LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute ß-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.