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11.
CtIP是DNA双链断裂修复中的关键蛋白之一,它能够促进断裂DNA末端切割,并且是一种已知的抑癌基因,与许多参与癌变过程的蛋白质如BRCA1,Rb等相互作用。为了更好地理解CtIP的分子网络,我们用在线工具PrePPI预测CtIP相互作用蛋白,发现PLK1是新的CtIP相互作用蛋白。PLK1在有丝分裂和癌症进展中发挥重要作用。我们进一步通过免疫沉淀法验证了它们的相互作用。 结果显示PLK1与CtIP有较强相互作用。此外,还采用Frodock 2.0工具对接CtIP和PLK1之间的蛋白质相互作用。最后,免疫沉淀测定和免疫荧光染色结果显示这两种蛋白质之间的相互作用与DNA损伤相关。基于这些结果,我们提出CtIP-PLK1相互作用可能在DNA损伤反应以及其他生物过程中发挥重要作用。 相似文献
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Zeng-Fu Shang Wei Tan Xiao-Dan Liu Lan Yu Bing Li Ming Li Man Song Yu Wang Bei-Bei Xiao Cai-Gao Zhong Hua Guan Ping-Kun Zhou 《International journal of biological sciences》2015,11(9):1026-1035
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair. DNA-PKcs has also been shown recently functioning in mitotic regulation. Here, we report that DNA-PKcs negatively regulates the stability of Cyclin B1 protein through facilitating its ubiquitination mediated by Cdh1 / E 3 ubiquitin ligase APC/C pathway. Loss of DNA-PKcs causes abnormal accumulation of Cyclin B1 protein. Cyclin B1 degradation is delayed in DNA-PKcs-deficient cells as result of attenuated ubiquitination. The impact of DNA-PKcs on Cyclin B1 stability relies on its kinase activity. Our study further reveals that DNA-PKcs interacts with APC/C core component APC2 and its co-activator Cdh1. The destruction of Cdh1 is accelerated in the absence of DNA-PKcs. Moreover, overexpression of exogenous Cdh1 can reverse the increase of Cyclin B1 protein in DNA-PKcs-deficient cells. Thus, DNA-PKcs, in addition to its direct role in DNA damage repair, functions in mitotic progression at least partially through regulating the stability of Cyclin B1 protein. 相似文献
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4E-BP1 participates in maintaining spindle integrity and genomic stability via interacting with PLK1
Zeng-Fu Shang Lan Yu Bing Li Wen-Zhi Tu Yu Wang Xiao-Dan Liu Hua Guan Bo Huang Wei-Qing Rang Ping-Kun Zhou 《Cell cycle (Georgetown, Tex.)》2012,11(18):3463-3471
The essential function of eIF4E-binding protein 1 (4E-BP1) in translation initiation has been well established; however, the role of 4E-BP1 in normal cell cycle progression is coming to attention. Here, we revealed the role of 4E-BP1 on mitotic regulation and chromosomal DNA dynamics during mitosis. First, we have observed the co-localization of the phosphorylated 4E-BP1 at T37/46 with Polo-like kinase 1 (PLK1) at the centrosomes during. Depression of 4E-BP1 by small interfering RNA in HepG2 or HeLa cells resulted in an increased outcome of polyploidy and aberrant mitosis, including chromosomal DNA misaligned and multi-polar spindles or multiple centrosomes. We observed that 4E-BP1 interacted with PLK1 directly in vitro and in vivo in mitotic cells, and the C-terminal aa 77–118 of 4E-BP1 mediates its interaction with PLK1. PLK1 can phosphorylate 4E-BP1 in vitro. Furthermore, the depletion of 4E-BP1 sensitized HepG2 and HeLa cells to the microtubule disruption agent paclitaxel. These results demonstrate that 4E-BP1, beyond its role in translation regulation, can function as a regulator of mitosis via interacting with PLK1, and possibly plays a role in genomic stability maintaining. 相似文献
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6-溴异香兰素BVAN08诱发肝癌细胞HepG2纺锤体损伤和有丝分裂灾变死亡 总被引:1,自引:0,他引:1
研究香兰素衍生物中的6.溴异香兰素(BVAN08)对细胞纺锤体结构的影响及诱发灾变死亡的相关机制,为开发该化合物为新的抗癌药物提供理论依据.通过光学显微镜观察BvAN08作用后细胞形态学变化,流式细胞术检测细胞周期,纺锤体功能检测点实验和原位免疫荧光杂交实验分析细胞有丝分裂进程和纺锤体结构.western印记检测BVAN08作用后相关蛋白质的变化.结果表明20~60 μmol/L BVAN08作用后,HepG2细胞变圆不再贴壁生长、随后脱落死亡,具有浓度依赖性量效关系;明显诱导细胞G2/M期阻滞、导致细胞有丝分裂指数升高,并出现大量的非二倍体和多倍体细胞;破坏细胞纺锤体的结构,多中心体细胞显著增加;该化合物促使细胞周期转录调节因子FoxM1及其下游靶分子细胞周期蛋白B1和CdK1的降解、阻止有丝分裂过程而导致有丝分裂灾变死亡.研究揭示BVAN08通过破坏纺锤体结构、诱发M期阻滞,导致细胞有丝分裂灾变死亡,FoxM1失活可能参与其作用机制. 相似文献
16.
Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent 总被引:3,自引:0,他引:3
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Hsieh PK Chang SC Huang CC Lee TT Hsiao CW Kou YH Chen IY Chang CK Huang TH Chang MF 《Journal of virology》2005,79(22):13848-13855
The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV. 相似文献
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Ying Chen Cui-Zhen Jin Xue-Qing Zhang Shi-Li Ge Ze-Yun Zhang Hui Xu Xiu-Lin Liu De-Chang Wu Ping-Kun Zhou 《Radiation and environmental biophysics》2009,48(1):57-65
On 25 June 1990, a radiation accident occurred in a 60Co source radiation unit in Shanghai, due to violations in operation regulations. This accident resulted in the exposure of
seven individuals to acute high-dose and dose-rate whole-body external irradiation. Conventional chromosomal aberration analysis,
G-banding automatic karyotype analysis and/or fluorescent in situ hybridization (FISH) painting methods were used to analyze
chromosomal aberrations in peripheral blood lymphocytes from five of the victims 24 h to 17 years after accidental exposure
to 1.9–5.1 Gy of 60Co γ-rays. The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at constant levels 1 month after
exposure. Three months after exposure, the frequency was reduced by 20–40% in three victims, while no reduction was seen in
the other two victims. Twelve years after exposure, the number of dicentrics and rings decreased by more than 90%, and did
not reveal a dose-dependent relationship. However, even at 12–17 years after exposure, stable chromosome aberrations, dominated
by translocations, remained at a high level in a dose-dependent manner. The frequency of stable chromosomal aberrations detected
by FISH showed a similar dose-dependent relationship as that detected by karyotype analysis of G-banding chromosomes. The
G-banding analysis also suggested that the pattern of chromosome breakpoints is random. The FISH data showed a decreasing
tendency with time for chromosome translocation frequency in the peripheral lymphocytes, and the rate of reduction varied
among different individuals. It is likely that the higher dose the victim received, the lesser the translocation frequency
decreased with time. The G-banding data also showed that the rate of reduction of translocations is different among individuals.
From 5 to 17 years after accidental irradiation, a very small reduction (~10%) of translocation frequency was observed in
victims C and D, while there was about a 35% reduction (the highest among the victims) for victim G who received the smallest
dose (1.9 Gy). These observations can be used to validate the existence of chromosomal aberrations in peripheral blood lymphocytes
as a biological dosimeter for radiation exposures. 相似文献
19.
c-Myc蛋白与DNA-PKcs作用位点的鉴定 总被引:1,自引:0,他引:1
DNA-PK复合物由Ku蛋白和DNA依赖蛋白激酶催化亚基(DNA-PKcs)组成,DNA-PKcs属于PI3K相关激酶家族成员.我们前期工作发现,DNA-Kcs沉默后,c-Myc的稳定性下降,且二者存在相互作用.为进一步确定c-Myc蛋白与DNA-PKcs相互作用位点,本研究利用原核表达系统活动了c-Myc及其截短体蛋白,利用GST pull-down技术结合Western印迹法,发现c-Myc蛋白294~370位氨基酸与DNA-PKcs存在相互作用.在细胞内表达GFP-c-Myc各截短体蛋白,发现294~370位氨基酸是c-Myc蛋白降解必需的.利用免疫荧光技术,发现DNA-PKcs与c-Myc蛋白有相同的细胞亚定位,进一步表明两者在生物学功能上具有相关性.有文献报道294~370位氨基酸是乙酰转移酶p300的底物,此位点的乙酰化导致c-Myc的降解.本实验结果提示,c-Myc蛋白的294~370位氨基酸与DNA-PKcs结合,可能阻止了乙酰转移酶p300的结合,从而达到提高c-Myc蛋白稳定性的作用. 相似文献