RESUMO
Efficient trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to and from the cell surface is essential for maintaining channel density at the plasma membrane (PM) and ensuring proper physiological activity. The most common mutation, F508del, exhibits reduced surface expression and impaired function despite treatment with currently available pharmacological small molecules, called correctors. To gain more detailed insight into whether CFTR enters compartments that allow corrector stabilization in the cell periphery, we investigated the peripheral trafficking itineraries and kinetics of wild type (WT) and F508del in living cells using high-speed fluorescence microscopy together with fluorogen activating protein detection. We directly visualized internalization and accumulation of CFTR WT from the PM to a perinuclear compartment that colocalized with the endosomal recycling compartment (ERC) markers Rab11 and EHD1, reaching steady-state distribution by 25 minutes. Stimulation by protein kinase A (PKA) depleted this intracellular pool and redistributed CFTR channels to the cell surface, elicited by reduced endocytosis and active translocation to the PM. Corrector or temperature rescue of F508del also resulted in targeting to the ERC and exhibited subsequent PKA-stimulated trafficking to the PM. Corrector treatment (24 hours) led to persistent residence of F508del in the ERC, while thermally destabilized F508del was targeted to lysosomal compartments by 3 hours. Acute addition of individual correctors, C4 or C18, acted on peripheral trafficking steps to partially block lysosomal targeting of thermally destabilized F508del. Taken together, corrector treatment redirects F508del trafficking from a degradative pathway to a regulated recycling route, and proteins that mediate this process become potential targets for improving the efficacy of current and future correctors.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/genética , Endocitose/fisiologia , Endossomos/genética , Endossomos/metabolismo , Endossomos/fisiologia , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/fisiologia , Microscopia de Fluorescência/métodos , Transporte Proteico , Proteólise , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Double B-box 1a (DBB1a) belongs to the zinc-finger family proteins in Arabidopsis thaliana. Transcriptional analysis uncovered that the DBB1a gene expression was blue light-dependently regulated, and the transcript level of DBB1a in cry1cry2 was decreased but not in phyAphyB compared to wild type under blue light conditions. Transgenic plants containing pDBB1a:GUS (ß-glucuronidase) displayed GUS activity in the vascular system of leaves and petioles. Green fluorescent protein (GFP)-fused DDB1a (DBB1a-GFP) protein was found in the nucleus in transient transformation assays with onion epidermal cells as well as in stable transgenic Arabidopsis plants. To investigate the function of DBB1a, we generated DBB1a over-expressing and under-expressing transgenic Arabidopsis plants. Analysis of hypocotyl growth of these lines indicated that DBB1a promoted hypocotyl elongation under blue light condition. The phenotype of transgenic plants with DBB1a over-expression could be impaired by a gibberellin (GA)-biosynthesis inhibitor. Moreover, the expression analysis of GA metabolic and catabolic genes in DBB1a transgenic lines indicated that the DBB1a suppressed GA2-oxidase1 (GA2ox1) and GA2-oxidase8 (GA2ox8) expression, but induced GA3ß-hydroxygenase1 (GA3ox1) and GA20-oxidase1 (GA20ox1) expression under blue light. Taken together, we concluded that DBB1a promotes hypocotyl elongation under blue light condition through an increase in bioactive GA levels in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Transporte/metabolismo , Giberelinas/metabolismo , Homeostase/efeitos da radiação , Hipocótilo/crescimento & desenvolvimento , Luz , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Criptocromos/metabolismo , Proteínas de Ligação a DNA , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/genética , Hipocótilo/efeitos da radiação , Mutação/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos da radiação , Feixe Vascular de Plantas/metabolismo , Feixe Vascular de Plantas/efeitos da radiação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição , Transcrição Gênica/efeitos da radiaçãoRESUMO
We report a photochemical synthesis of Pd/CeO2 catalysts with atomically dispersed Pd. Compared to atomically dispersed Pd/CeO2 with a cubic CeO2 support (Pd/CeO2-CP), atomically dispersed Pd/CeO2 with a truncated octahedral CeO2 support (Pd/CeO2-TOP) exhibited higher activity and selectivity, owing to the synergy between Pd atoms and the (111) surface of CeO2. When compared to Pd/CeO2 with Pd clusters and nanoparticles via chemical reduction, Pd/CeO2-TOP showed excellent activity with an enhancement factor of 324 in CO oxidation, as well as an activity enhancement by a factor of 344 in selective oxidation of benzyl alcohol.
RESUMO
Hepatocyte nuclear factor-1beta plays an important role in the development and progression of liver cancer. In recent years, the expression of HNF-1ß has been reported to be associated with risk for a variety of cancers. The purpose of this study is to investigate whether the expression of HNF-1ß promotes the malignancy of HCC and its mechanism. We retrospectively investigated the expression of HNF-1ß in 90 patients with hepatocellular carcinoma and found that the high expression of HNF-1ß indicated poor prognosis. We overexpressed HNF-1ß in liver cancer cell lines and found the expression of liver progenitor cell markers and stemness were upregulated. The invasion ability and epithelial-mesenchymal transition (EMT)-associated genes were also significantly higher in liver cancer cells overexpressing HNF-1ß than in the control group. A mechanistic study suggested the activation of the Notch signalling pathway probably plays a key role downstream of HNF-1ß. More importantly, HNF-1ß promoted tumourigenesis of HCC cells in vivo. In conclusion, high expression of HNF-1ß not only promoted the de-differentiation of HCC cells into liver cancer stem cells through activating the Notch pathway but also enhanced the invasive potential of HCC cells and EMT occurrence, which would contribute to the enhancement of cell migration and invasion.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Fator 1-beta Nuclear de Hepatócito/genética , Neoplasias Hepáticas/genética , Células-Tronco Neoplásicas/metabolismo , Receptor Notch1/genética , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Desdiferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Hepatectomia , Fator 1-beta Nuclear de Hepatócito/metabolismo , Xenoenxertos , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Receptor Notch1/metabolismo , Estudos Retrospectivos , Transdução de Sinais , Análise de SobrevidaRESUMO
Cryptochromes are blue-light photoreceptors that control many aspects of plant development. In this study, cryptochrome mutants of Arabidopsis were examined to assess the role of cryptchrome-1 (CRY1) in lateral roots growth. When grown in blue light for 12d, mutant seedlings (cry1) showed increased growth of lateral roots, while CRY1-overexpressing transgenic seedlings (CRY1ox) exhibited a marked decrease. Lateral roots growth of CRY1ox could be stimulated by auxin, but expression of PIN1 (efflux carrier of polar auxin transport) was strongly reduced. Contrary, the cry1 mutation showed the opposite effect, indicating that blue light and the auxin-signaling pathway interact in lateral roots growth of Arabidopsis. The free IAA content in CRY1ox roots was half of that in wild type and cry1 mutant roots. Moreover, the content of flavonoids (quercetin, kaempferol, isorhamnetin), which act as endogenous negative regulators of auxin transport, increased in CRY1ox seedlings. Taken together, these results suggest that Arabidopsis CRY1 restrains lateral roots growth by inhibiting auxin transport.