RESUMO
NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system.
Assuntos
Apoptose , Receptores ErbB/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Musculares/citologia , Células Musculares/enzimologia , Músculo Esquelético/citologia , Neuraminidase/metabolismo , Animais , Western Blotting , Caspases/metabolismo , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Citoproteção , Ativação Enzimática , Gangliosídeo G(M3)/metabolismo , Inativação Gênica , Camundongos , Modelos Biológicos , Sialiltransferases/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Esfingolipídeos/metabolismo , Regulação para Cima/genéticaRESUMO
The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.
Assuntos
Antineoplásicos/farmacologia , Morfolinas/farmacologia , Purinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Benzotiazóis/farmacologia , Western Blotting , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular , Morte Celular , Desdiferenciação Celular , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular , Endorreduplicação , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Citometria de Fluxo , Inativação Gênica , Células HeLa , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography-mass spectrometry (GLC-MS) analysis of 1,7-sialolactones in biological media.
Assuntos
Lactonas/síntese química , Ácidos Siálicos/síntese química , Configuração de Carboidratos , Lactonas/química , Ácidos Siálicos/química , EstereoisomerismoRESUMO
Work is presented on the role of cAMP-dependent protein phosphorylation in post-translational processing and biosynthesis of complex I subunits in mammalian cell cultures. PKA-mediated phosphorylation of the NDUFS4 subunit of complex I promotes in cell cultures in vivo import/maturation in mitochondria of the precursor of this protein. The import promotion appears to be associated with the observed cAMP-dependent stimulation of the catalytic activity of complex I. These effects of PKA are counteracted by activation of protein phosphatase(s). PKA and the transcription factor CREB play a critical role in the biosynthesis of complex I subunits. CREB phosphorylation, by PKA and/or CaMKs, activates at nuclear and mitochondrial level a transcriptional regulatory cascade which promotes the concerted expression of nuclear and mitochondrial encoded subunits of complex I and other respiratory chain proteins.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Complexo I de Transporte de Elétrons/genética , Humanos , Técnicas In Vitro , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , NADH Desidrogenase/química , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , RatosRESUMO
In this paper the regulatory features of complex I of mammalian and human mitochondria are reviewed. In a variety of mitotic cell-line cultures, activation in vivo of the cAMP cascade, or direct addition of cAMP, promotes the NADH-ubiquinone oxidoreductase activity of complex I and lower the cellular level of ROS. These effects of cAMP are found to be associated with PKA-mediated serine phosphorylation in the conserved C-terminus of the subunit of complex I encoded by the nuclear gene NDUFS4. PKA mediated phosphorylation of this Ser in the C-terminus of the protein promotes its mitochondrial import and maturation. Mass-spectrometry analysis of the phosphorylation pattern of complex I subunits is also reviewed.
