背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚...背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚型之一的AGPAT5在癌症中的研究还十分有限,本研究深入剖析AGPAT5在肝癌发生、发展中发挥的作用及潜在的分子机制,旨在为肝癌诊断和治疗策略提供新思路。方法:利用慢病毒感染将多种肝癌细胞系中的AGPAT5敲减,并通过锥虫蓝计数、划痕、transwell及平板克隆等实验在体外检测AGPAT5对肝癌细胞增殖、迁移及抗失巢凋亡能力的影响。通过回复野生型或酶活性缺失型的AGPAT5,探究其作为代谢酶是否发挥经典代谢作用调控肝癌细胞迁移。构建BALB/c裸鼠尾静脉注射移植瘤模型,从体内层面验证体外的细胞表型。采用免疫沉淀质谱联用(immunoprecipitation mass spectrum,IP-MS)鉴定出与AGPAT5相互作用的蛋白,并进行免疫共沉淀(co-immunoprecipitation,coIP)验证。蛋白质翻译后通过修饰鉴定分析AGPAT5潜在的修饰位点,通过体外实验探究点突变前后对肝癌细胞迁移的影响。通过coIP探究该位点突变前后AGPAT5与相互作用蛋白结合的情况。通过敲低相互作用蛋白确定其在细胞表型中的作用。通过回复实验验证AGPAT5是否通过相互作用蛋白发挥作用。检测野生型肝癌细胞系中的AGPAT5和相互作用蛋白的表达水平,检验两者之间是否具有相关性。结果:肝癌细胞敲减AGPAT5后会更加耐受无血清饥饿,并促进细胞迁移,但不会影响细胞增殖和失巢凋亡。而酶活性缺失并不影响AGPAT5对肝癌细胞迁移的抑制。敲减AGPAT5可促进肝癌细胞在裸鼠体内的肺转移和肝转移。AGPAT5可以与原纤维蛋白(fibrillarin,FBL)相互作用,并在无血清饥饿刺激下加强两者的结合。遏制FBL的表达会抑制肝癌细胞迁移,且效果与过表达AGPAT5相似。抑制FBL的表达可削弱敲低AGPAT5对肝癌细胞迁移的促进作用。在已检测的肝癌细胞系中,AGPAT5和FBL在蛋白水平上并不存在相关性。K201位点突变使AGPAT5对肝癌细胞迁移的抑制作用减弱,并使AGPAT5与FBL的结合减弱。结论:敲低AGPAT5能够显著提高肝癌细胞迁移能力。AGPAT5可以与FBL相互作用,在无血清饥饿刺激下,AGPAT5或通过K201位点的乙酰化加强与FBL的结合,从而更有效地遏制FBL,进而抑制肝癌细胞迁移。但这种抑制作用并非来自AGPAT5的代谢酶活性,而是由非代谢作用所驱动。展开更多
Fibrillann, a major protein in the nucleolus. is known to redistribute during mitosis from the nucleolus to the cytosol, and is related to the dynamics of post-mitotic reassembly of the nucleolus. To better understand...Fibrillann, a major protein in the nucleolus. is known to redistribute during mitosis from the nucleolus to the cytosol, and is related to the dynamics of post-mitotic reassembly of the nucleolus. To better understand the dynamic behavior and the relationship with other cytoplasmic structures, we have now expressed fibrillarin-pDsRedl fusion protein in HeLa cells. The results showed that a part of fibrillarin was associated with mitotic spindle poles in the mitotic cells. Nocodazole-induced microtubule depolymeri-zation resulted in fibrillarin redistribution throughout the cytoplasm, and removal of nocodazole resulted in relocaliza-tion of fibrillarin at the polar region during the mitotic spindles reassembly. In a mitotic cell free system, fibrillarin was found in the center of taxol-induced microtubule asters. Moreover, fibrillarin was found to colocalize with the nuclear mitotic apparatus protein (NuMA) at the poles of mitotic cells. Therefore, it is postulated that the polar redistribution of展开更多
Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown a...Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6-7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fib-rillarin are two major protein components of nucleolar skeleton of HeLa cells.展开更多
Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific ...Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.展开更多
文摘背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚型之一的AGPAT5在癌症中的研究还十分有限,本研究深入剖析AGPAT5在肝癌发生、发展中发挥的作用及潜在的分子机制,旨在为肝癌诊断和治疗策略提供新思路。方法:利用慢病毒感染将多种肝癌细胞系中的AGPAT5敲减,并通过锥虫蓝计数、划痕、transwell及平板克隆等实验在体外检测AGPAT5对肝癌细胞增殖、迁移及抗失巢凋亡能力的影响。通过回复野生型或酶活性缺失型的AGPAT5,探究其作为代谢酶是否发挥经典代谢作用调控肝癌细胞迁移。构建BALB/c裸鼠尾静脉注射移植瘤模型,从体内层面验证体外的细胞表型。采用免疫沉淀质谱联用(immunoprecipitation mass spectrum,IP-MS)鉴定出与AGPAT5相互作用的蛋白,并进行免疫共沉淀(co-immunoprecipitation,coIP)验证。蛋白质翻译后通过修饰鉴定分析AGPAT5潜在的修饰位点,通过体外实验探究点突变前后对肝癌细胞迁移的影响。通过coIP探究该位点突变前后AGPAT5与相互作用蛋白结合的情况。通过敲低相互作用蛋白确定其在细胞表型中的作用。通过回复实验验证AGPAT5是否通过相互作用蛋白发挥作用。检测野生型肝癌细胞系中的AGPAT5和相互作用蛋白的表达水平,检验两者之间是否具有相关性。结果:肝癌细胞敲减AGPAT5后会更加耐受无血清饥饿,并促进细胞迁移,但不会影响细胞增殖和失巢凋亡。而酶活性缺失并不影响AGPAT5对肝癌细胞迁移的抑制。敲减AGPAT5可促进肝癌细胞在裸鼠体内的肺转移和肝转移。AGPAT5可以与原纤维蛋白(fibrillarin,FBL)相互作用,并在无血清饥饿刺激下加强两者的结合。遏制FBL的表达会抑制肝癌细胞迁移,且效果与过表达AGPAT5相似。抑制FBL的表达可削弱敲低AGPAT5对肝癌细胞迁移的促进作用。在已检测的肝癌细胞系中,AGPAT5和FBL在蛋白水平上并不存在相关性。K201位点突变使AGPAT5对肝癌细胞迁移的抑制作用减弱,并使AGPAT5与FBL的结合减弱。结论:敲低AGPAT5能够显著提高肝癌细胞迁移能力。AGPAT5可以与FBL相互作用,在无血清饥饿刺激下,AGPAT5或通过K201位点的乙酰化加强与FBL的结合,从而更有效地遏制FBL,进而抑制肝癌细胞迁移。但这种抑制作用并非来自AGPAT5的代谢酶活性,而是由非代谢作用所驱动。
基金This work was supported by the State Key Basic Research Program of China (Grant No. G1999053906).
文摘Fibrillann, a major protein in the nucleolus. is known to redistribute during mitosis from the nucleolus to the cytosol, and is related to the dynamics of post-mitotic reassembly of the nucleolus. To better understand the dynamic behavior and the relationship with other cytoplasmic structures, we have now expressed fibrillarin-pDsRedl fusion protein in HeLa cells. The results showed that a part of fibrillarin was associated with mitotic spindle poles in the mitotic cells. Nocodazole-induced microtubule depolymeri-zation resulted in fibrillarin redistribution throughout the cytoplasm, and removal of nocodazole resulted in relocaliza-tion of fibrillarin at the polar region during the mitotic spindles reassembly. In a mitotic cell free system, fibrillarin was found in the center of taxol-induced microtubule asters. Moreover, fibrillarin was found to colocalize with the nuclear mitotic apparatus protein (NuMA) at the poles of mitotic cells. Therefore, it is postulated that the polar redistribution of
文摘Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6-7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fib-rillarin are two major protein components of nucleolar skeleton of HeLa cells.
基金Acknowledgments We thank the cell biology core facility for confocal study. The PHF8 antibody was kindly provided by Dr Jiemin Wong （East China Normal University）. This work was supported by the National Basic Research Program of China （2007CB947900, 2010CB529705, 2007CB947100）, the Chinese Academy of Sci- ences （KSCX2-YW-R-04, KSCX2-YW-R-I 11）, the National Natural Science Foundation of China （30870538, 90919026）, Postdoctoral fellowship （20090460670）, and the Council of Shanghai Municipal Government for Science and Technology.
文摘Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.
