高迁移率族蛋白B1(high mobility group box-1,HMGB1)是一种在哺乳动物中广泛存在、进化中高度保守的蛋白质。细胞核定位序列的存在使得生理状态下的HMGB1主要存在于细胞核内,但其在细胞内外的不同位置具有不同的生物学功能。细胞外的HM...高迁移率族蛋白B1(high mobility group box-1,HMGB1)是一种在哺乳动物中广泛存在、进化中高度保守的蛋白质。细胞核定位序列的存在使得生理状态下的HMGB1主要存在于细胞核内,但其在细胞内外的不同位置具有不同的生物学功能。细胞外的HMGB1与脓毒症、肿瘤、风湿免疫、动脉粥样硬化及缺血再灌注损伤等多种疾病密切相关。HMGB1由细胞核内迁移至细胞质再释放到细胞外的过程,涉及核定位序列的翻译后修饰、主动分泌或被动释放等机制。展开更多
Objective: To investigate the functions of nM-CSF in malignant cells. Methods: recombinant M-CSF was targeted into cell nucleus by employing a eukaryotic expression plasmid vector pCMV/myc/nuc. The constructed plasmid...Objective: To investigate the functions of nM-CSF in malignant cells. Methods: recombinant M-CSF was targeted into cell nucleus by employing a eukaryotic expression plasmid vector pCMV/myc/nuc. The constructed plasmid was transfected into cells of EBV transformed lymphoblastoid cell line (LCL). RT-PCR, Western blot and immunofluorescent staining showed that recombinant M-CSF was localized into LCL cell nucleus. The transgenic cells showed elevated proliferation potential, enhanced resistance to apoptosis and increased ability of in vitro migration. Conclusion: Nucleus presenting M-CSF might act as a promoting factor in the processes of cell malignancy.展开更多
Tip60 is a specific member of MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of nuclear histone acetyltransferases (HAT). It is essential for cellular survival, differentiation, and metabolism. A putative canonical NLS motif ...Tip60 is a specific member of MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of nuclear histone acetyltransferases (HAT). It is essential for cellular survival, differentiation, and metabolism. A putative canonical NLS motif between the chromo domain and the zinc finger of Tip60 was identified. Here we show evidence that Tip60 is associated with importin α as its substrate and transported from cytoplasm to the nucleus. Pull down assay revealed that Tip60 was physically associated with importin α both in vivo and in vitro. Confocal microscopic observation showed that Tip60 and importin α were co-localized with each other. The localization of Tip60 to the nuclear and its interaction with importin α was disrupted when its putative NLS motif for binding to importin α was mutated (219RKRK222 → 219AAAA222). However, attachment of this putative NLS motif to a cytoplasmic protein (YAP 1-210 fragment) promoted its nuclear localization. Based on transient transfection, Tip60 NLS motif mutant showed a substantial reduction in self-acetylation, HAT activity, and apoptotic ability whereas wild type Tip60 did not show such reduction. Taken together, our results demonstrate that importin α transports Tip60 from the cytoplasm to the nucleus through binding to the putative NLS motif of Tip60 for its tumor suppressing function.展开更多
Objective: To explore the effects of nuclear M-CSF on the process of tumorigenesis. Methods: Functional part of M-CSF cDNA was inserted into an eukaryotic expression plasmid pCMV/myc/nuc, which can add three NLS to t...Objective: To explore the effects of nuclear M-CSF on the process of tumorigenesis. Methods: Functional part of M-CSF cDNA was inserted into an eukaryotic expression plasmid pCMV/myc/nuc, which can add three NLS to the C-terminal of the expressed protein and direct the protein into the cell nuclei. The constructed plasmid was transferred into NIH3T3 cells and the cell clones were selected by G-418 selection. Cell clones stable expressing target protein were identified by RT-PCR, ABC immunohistochemistry assayand Western blot. Cell growth kinetics analyses throughgrowth curves, cell doubling time, MTT test and anti-sense oligodeoxynucleotide (ASODN) inhibiting cell growth testwere performed to identify cells proliferation potential.Results: The transfected cells showed elevated proliferation potential over the control cells. Conclusion: Abnormalappearance of M-CSF in nucleus could enhance cellproliferation, which suggests that cytokine isoforms within cell nucleus might play transcription factor-like role.展开更多
文摘高迁移率族蛋白B1(high mobility group box-1,HMGB1)是一种在哺乳动物中广泛存在、进化中高度保守的蛋白质。细胞核定位序列的存在使得生理状态下的HMGB1主要存在于细胞核内,但其在细胞内外的不同位置具有不同的生物学功能。细胞外的HMGB1与脓毒症、肿瘤、风湿免疫、动脉粥样硬化及缺血再灌注损伤等多种疾病密切相关。HMGB1由细胞核内迁移至细胞质再释放到细胞外的过程,涉及核定位序列的翻译后修饰、主动分泌或被动释放等机制。
基金This work was supported by the Climbing Program Granted by the Ministry of Science and Technology of China(95-special-10) and Tianjin Science and TechnologyDevelopment Project (No. 003119311).
文摘Objective: To investigate the functions of nM-CSF in malignant cells. Methods: recombinant M-CSF was targeted into cell nucleus by employing a eukaryotic expression plasmid vector pCMV/myc/nuc. The constructed plasmid was transfected into cells of EBV transformed lymphoblastoid cell line (LCL). RT-PCR, Western blot and immunofluorescent staining showed that recombinant M-CSF was localized into LCL cell nucleus. The transgenic cells showed elevated proliferation potential, enhanced resistance to apoptosis and increased ability of in vitro migration. Conclusion: Nucleus presenting M-CSF might act as a promoting factor in the processes of cell malignancy.
文摘Tip60 is a specific member of MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of nuclear histone acetyltransferases (HAT). It is essential for cellular survival, differentiation, and metabolism. A putative canonical NLS motif between the chromo domain and the zinc finger of Tip60 was identified. Here we show evidence that Tip60 is associated with importin α as its substrate and transported from cytoplasm to the nucleus. Pull down assay revealed that Tip60 was physically associated with importin α both in vivo and in vitro. Confocal microscopic observation showed that Tip60 and importin α were co-localized with each other. The localization of Tip60 to the nuclear and its interaction with importin α was disrupted when its putative NLS motif for binding to importin α was mutated (219RKRK222 → 219AAAA222). However, attachment of this putative NLS motif to a cytoplasmic protein (YAP 1-210 fragment) promoted its nuclear localization. Based on transient transfection, Tip60 NLS motif mutant showed a substantial reduction in self-acetylation, HAT activity, and apoptotic ability whereas wild type Tip60 did not show such reduction. Taken together, our results demonstrate that importin α transports Tip60 from the cytoplasm to the nucleus through binding to the putative NLS motif of Tip60 for its tumor suppressing function.
基金This work was supported by a grant from Tianjin Science and Technology Development Project (No. 003119311).
文摘Objective: To explore the effects of nuclear M-CSF on the process of tumorigenesis. Methods: Functional part of M-CSF cDNA was inserted into an eukaryotic expression plasmid pCMV/myc/nuc, which can add three NLS to the C-terminal of the expressed protein and direct the protein into the cell nuclei. The constructed plasmid was transferred into NIH3T3 cells and the cell clones were selected by G-418 selection. Cell clones stable expressing target protein were identified by RT-PCR, ABC immunohistochemistry assayand Western blot. Cell growth kinetics analyses throughgrowth curves, cell doubling time, MTT test and anti-sense oligodeoxynucleotide (ASODN) inhibiting cell growth testwere performed to identify cells proliferation potential.Results: The transfected cells showed elevated proliferation potential over the control cells. Conclusion: Abnormalappearance of M-CSF in nucleus could enhance cellproliferation, which suggests that cytokine isoforms within cell nucleus might play transcription factor-like role.
