RESUMO
PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process.
Assuntos
Bass , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , PTEN Fosfo-Hidrolase/genética , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência/veterinária , Distribuição Tecidual , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio alginolyticus/fisiologiaRESUMO
Bcl-2 is a pro-survival member of Bcl-2 like superfamily, playing an important role in regulating the apoptotic process. In this study, the full-length Bcl-2 (EcBcl-2) was obtained, consisting of a 5'UTR of 290 bp, an ORF of 699 bp and a 3'UTR of 920 bp. EcBcl-2 gene encoded a polypeptide of 232 amino acids with an estimated molecular mass of 26.12 KDa and a predicted isoelectric point (pI) of 6.93. The deduced amino acid sequence analysis showed that EcBcl-2 consisted of the conserved residues and characteristic domains known to the critical functionality for Bcl-2. qRT-PCR analysis revealed that EcBcl-2 transcript was expressed in all the examined tissues, while the strongest expression level was observed in liver, followed by the expression in blood, gill, kidney, spleen, heart, intestine and muscle. The groupers challenged with V. alginolyticus showed a significant increase of EcBcl-2 mRNA in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcBcl-2 protein expression was detected in liver. Subcellular localization analysis revealed that EcBcl-2 was localized in both nucleus and cytoplasm. Overexpression of EcBcl-2 can inhibit the LPS-induced apoptosis and activate the transcription activity of NF-κB and AP-1, while the deletion of BH1, BH2, BH3 or BH4 domain from EcBcl-2 can impede the signaling transduction. These results indicate that EcBcl-2 may play a regulatory role in the apoptotic process.
Assuntos
Bass/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/genética , Clonagem Molecular/métodos , Citoplasma/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , NF-kappa B/genética , RNA Mensageiro/genética , Alinhamento de Sequência , Fator de Transcrição AP-1/genética , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
Bax is a pro-apoptotic member of Bcl-2 like superfamily, playing an important role in regulating the apoptosis. In this study, the full-length Bax (EcBax) was obtained, containing a 5'UTR of 64 bp, an ORF of 579 bp and a 3'UTR of 1021 bp. The EcBax gene encoded a polypeptide of 192 amino acids with an estimated molecular mass of 21.55 KDa and a predicted isoelectric point (pI) of 6.75. The deduced amino acid sequence analysis showed that EcBax comprised the conserved residues and the characteristic domains known to the critical function of Bax. qRT-PCR analysis revealed that EcBax mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in blood, followed by the expression in liver, gill, spleen, kidney, heart, muscle and intestine. A sharp increase of EcBax expression was observed in the vibrio challenge group by comparing with those in the control. Subcellular localization analysis revealed that EcBax was predominantly localized in the cytoplasm. EcBax exerted a regulatory role in modulating the mitochondrial membrane potential, promoting the cytochrome c release, and then activating the downstream caspase signaling. Moreover, the overexpression of EcBax can decrease the cell viability and antagonize NF-kB, AP-1, Stat3 promoter activity in Hela cells. These results indicate that EcBax containing the conserved domain of pro-apoptotic member of Bcl-2 family may disrupt the mammalian signaling and play a regulative role in the apoptotic process.