HTLV—1转录激活因子Tax和Taxreb107的相互作用

Tax是人类T淋巴细胞白血病病毒编码的转录因子。核糖体蛋白RpL6又称Taxreb10 7,是Tax应答序列结合蛋白 ,二者均作用于HTLV 1的启动子LTR。用酵母双杂交法和GST下拉检测 (pull down)法研究了Tax和Taxreb10 7/RpL6之间的相互关系。结果显示 ,二者在酵母细胞内和体外均具有直接相互作用。这些结果提示Taxreb10 7/RpL6可能通过与Tax的相互作用而调节Tax在病毒感染中的作用     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

猪流行性腹泻病毒核衣壳蛋白与感染细胞核磷蛋白的共定位分析

【目的】阐明猪流行性腹泻病毒(PEDV)核衣壳蛋白与病毒感染细胞核仁成分B23.1蛋白的共定位特征。【方法】分别参照GenBank中PEDV CV777株的N基因序列(AF353511)和编码人细胞核仁蛋白B23.1基因序列(BC050628.1),设计、合成扩增N基因和B23.1基因的引物,利用RT-PCR技术扩增了N基因和Vero E6细胞的B23.1基因的cDNA,分别克隆到真核表达载体pAcGFP1-C1和pDsRed2-N1,获得重组质粒pAcGFP1-C1/N和pDsRed2-N1/B23.1,共转染Vero E6细胞。【结果】Western blots分析表明这些融合蛋白在转染的Vero E6细胞中表达;共聚焦显微镜技术分析表明在共转染Vero E6细胞中猪流行性腹泻病毒N蛋白与Vero E6细胞核磷蛋白B23.1发生共定位。【结论】为进一步鉴定PEDV N蛋白中核仁定位信号和N蛋白核仁定位机制提供可靠依据。     

核定位信号在热休克蛋白 70 抑制氧化应激所致 细胞核仁分离中的作用

为探讨核定位信号在热休克蛋白 70 (HSP70) 抑制 H²O² 所致核仁损伤中的作用,采用分子克隆技术分别构建了 4 个真核表达载体, pcDNA3.1(-)-HSP70^WT (HSP70 野生型), pcDNA3.1(-)-HSP70^ΔNLS (核定位信号缺失突变体 ), pEGFP-N1-HSP70^WT, pEGFP-N1- HSP70^ΔNLS. 向传代培养的 C²C¹²肌源细胞培养液中加入终浓度为 1.0 mmol/L 的 H²O² 模拟体外氧化应激 . 甲苯胺蓝染色细胞核仁发现,正常细胞仅有一个位于中央的、浓染致密的核仁颗粒 . 过氧化氢处理后 3 h ,可见明显的核仁分离 . 热休克反应处理的细胞及转 pcDNA3.1( － )-HSP70WT 细胞则能明显抑制氧化应激所致的核仁分离 . 荧光蛋白示踪及核仁蛋白质免疫印迹分析显示, H²O²处理后 1 h , HSP70WT 由正常时的细胞浆定位转为细胞核及核仁定位,而 HSP70ΔNLS 在 H²O²处理后仍定位于细胞浆,同时丧失了抑制核仁分离的作用 . 上述结果提示,野生型 HSP70 能显著抑制氧化应激所致细胞核仁分离,核定位信号通过介导 HSP70 向细胞核及核仁移位而决定 HSP70 对核仁损伤的保护作用 .     

多肽TAT与核定位信号介导的蛋白质入核递送

增强型绿色荧光蛋白与蛋白质转导结构域TAT、SV40大T抗原的核定位信号以融合蛋白的形式在大肠杆菌中表达 ,纯化后转导A431细胞 ,大部分细胞核内都可以观察到绿色荧光 ,说明TAT NLS可以有效介导蛋白质的入核递送。这种蛋白质递送系统可望用于转录治疗等研究领域。     

核定位信号及其分析策略

真核细胞核膜上的核孔复合体 (nuclear pore complex, NPC) 是细胞核内外进行物质交换的主要通道, 分子量较小的化合物可自由通过NPC或采取被动扩散的方式进入细胞核, 而分子量为50 kD以上的蛋白质则只能通过主动转运进入细胞核. 以这种方式进入细胞核的 蛋白质必须在其氨基酸序列上拥有特殊的核定位信号(nuclear localization signal, NLS)以被相应的核转运蛋白(karyopherins) 识别. 核定位信号具有多样性, 包括经典核定位信号(classical NLS,cNLS), 内输蛋白β2识别的核定位信号（又称PY模体-NLS）和其它类型的NLS. 每一类NLS具有相似的特征, 但并不具有完全保守的氨基酸组成. 不同的NLS, 往往对应着各不相同的核输入机制. 而对同一蛋白质来说, 也可能同时拥有几个功能性的NLS. 研究核定位信号一方面可以帮助揭示新的大分子物质核转运机制, 另一方面也有助于发现一些蛋白质的新功能. 本文对常见NLS的分类进行了总结, 并介绍了两种常用的NLS预测软件及鉴定NLS的一般策略.     

核定位信号筛选系统的构建

建立了一酵母克隆系统用于克隆含核定位信号 (NLS)的蛋白质的基因 .用表达转录因子GAL4 DNA结合域 - p53(GAL4- DBD- p53)融合蛋白的质粒转化酵母 HF7c,使 GAL4- DBD- p53可结合于报告基因的启动子但因无转录激活域而不能激活转录 .构建一酵母穿梭载体 ,可表达无NLS的 GAL4转录激活域 -大 T抗原 (GAL4- AD- LT)融合蛋白 .融合蛋白基因的下游插入一多克隆位点 .将 c DNA文库插入多克隆位点后 ,如果 c DNA片段可编码 NLS,则 GAL4- AD- LT分子可进入细胞核 ,并通过 LT与 p53的相互作用而使 GAL4- AD结合于启动子和激活报告基因的转录 .构建了这一克隆系统的各质粒 ,并用绿色荧光蛋白 (GFP)验证了其对核内蛋白和胞浆蛋白的甄别能力 .这一系统将有助于从 c DNA文库中筛选编码带有 NLS的蛋白质的基因     

核糖体蛋白rpS6在核仁中的定位与其磷酸化无关

核糖体蛋白S6(rpS6)是核糖体小亚基40S的一个组成成分。在该研究中,利用免疫荧光和邻位连接技术证明rpS6不仅是核糖体小亚基的组成成分,而且还可与核仁中的U3核蛋白复合体的标志性蛋白Mpp10共定位并且存在相互作用。rpS6蛋白的C端有5个丝氨酸磷酸化位点,为了研究rpS6蛋白在核仁中的分布是否与其磷酸化有关,构建了rpS6蛋白的两个突变体rpS6A和rpS6D分别与EGFP和HA的融合蛋白。rpS6A是将C端的5个丝氨酸位点全部突变为丙氨酸;rpS6D是将C端的5个丝氨酸位点全部突变为天冬氨酸。研究表明:rpS6、rpS6A和rpS6D与EGFP和HA的融合蛋白均可分布在核仁中,与内源性rpS6蛋白的分布情况一致,说明rpS6蛋白在核仁中的定位与其磷酸化无关,为探索rpS6蛋白在核仁中的功能奠定了良好的基础。     

蛋白质的入核运送和它在基因表达中的调节作用

蛋白质进入细胞核是由蛋白质分子内部的核定位信号（nuclear localization signal, NLS）引导的.NLS蛋白首先与NLS受体结合,然后在多种胞浆因子及核孔复合物蛋白的作用下穿过核孔、转位入核.蛋白质上存在NLS并不一定总能够引导蛋白质入核.当NLS被修饰或遮掩时,它们便不能被核转运装置所识别.因而,NLS的遮掩被解除之前,蛋白质一直被扣留在胞浆中.以调节转录因子的入核运送来控制转录因子的活性是基因表达调节的一个新概念,也是细胞生长和分化的另一水平的调节.     

植物细胞质介导GFP-NLS蛋白入核转运的HeLa细胞系统的建立北大核心CSCD

细胞核作为细胞中重要的遗传物质存储、复制和转录的结构,涉及大量信息和物质的传输活动,尤其是蛋白质的入核转运一直以来都是研究的热点问题之一。本研究证明,植物细胞质可以有效应用于动物细胞体系研究蛋白质入核转运。本文利用病毒SV40抗原蛋白中的核定位信号(nuclear localization signal,NLS)标记绿色荧光蛋白(green fluorescent protein,GFP),通过拟南芥细胞质的介导,利用He La细胞核建立研究蛋白质入核转运的半细胞体系。结果显示,植物细胞质结合NLS片段能改变GFP在He La细胞核内外的分布,实现对目标蛋白入核过程的介导,使GFP-NLS最后定位于细胞核内。这也意味着通过He La细胞建立起的半细胞体系能为蛋白质入核转运研究提供一个有效的研究体系。     

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response     

Translocation of fibroblast growth factor-10 and its receptor into nuclei of human urothelial cells

Fibroblast growth factor-10 (FGF-10), a mitogen for the epithelial cells lining the lower urinary tract, has been identified inside urothelial cells, despite its acknowledged role as an extracellular signaling ligand. Recombinant (r)FGF-10 was determined by fluorescence microscopy optical sectioning to localize strongly to nuclei inside cultured urothelial cells. To clarify the possible role of a nuclear localization signal (NLS) in this translocation, a variant of rFGF-10 was constructed which lacked this sequence. rFGF-10(no NLS) was found in cytoplasm to a far greater degree than rFGF-10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or non-existent bioactivity compared to the wild-type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF-10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF-10. The FGF-10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous rFGF-10 ligand. Co-localization studies between rFGF-10 and the FGF-10 receptor revealed a strong intracellular relationship between the two. This co-localization was seen in nuclei for both rFGF-10 and for rFGF-10(no NLS), although the correlation was weaker for rFGF-10(no NLS). These data show that an NLS-like motif of rFGF-10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF-10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue.     

Functional characterization of nuclear localization signals in yeast Sm proteins

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.     

PNRC1核定位信号序列的鉴定

为鉴定富含脯氨酸核受体辅调节蛋白1(PNRC1)分子的核定位信号序列（nuclear localization signal sequence, NLS）,在生物信息学方法预测的基础上,先构建野生型PNRC1及删除预测NLS的PNRC1突变体的绿色荧光蛋白（GFP）重组表达载体,转染细胞后通过激光共聚焦显微镜观察PNRC1分子在删除预测NLS后细胞内的定位变化.然后,将预测的NLS编码序列直接连到GFP表达载体上,以及将预测的NLS加到胞浆蛋白上构建其GFP重组表达载体,转染细胞,观察预测的NLS能否把构建的重组体都带到细胞核内.结果显示,删除PNRC1中预测的NLS后,其定位从细胞核中变为主要定位在细胞浆中,而预测的NLS能把GFP或胞浆中的蛋白带到细胞核中.研究表明,预测的NLS为PNRC1分子真正的NLS.     

Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

Porcine circovirus type 1 （PCV1） contains two major open reading frames encoding the replication-associated proteins and the major structural capsid （Cap） protein. PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals （NLS）. The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCVI Cap versions fused to enhanced green fluorescent protein （EGFP）. The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RR- RR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.     

Independent signals determine the subcellular localization of NEP in prostate cancer cells

NEP (Neutral endopeptidase 24.11) is a cell surface enzyme that hydrolyzes bioactive neuropeptides implicated in the transition from androgen-dependent prostate cancer (PC) to androgen-independent PC. We report the cloning and sequence analyses of NEP cDNAs from human androgen-responsive LNCaP PC cells and prostatic stromal cells. To investigate the functional role of a nuclear localization sequence (NLS) detected within the N-terminus and of an endoplasmic reticulum retention signal within the C-terminus, NEP-GFP expression vectors were constructed containing the whole NEP gene, fragments encoding the N-terminus/C-terminus of the protein (5(')NEP-GFP/3(')NEP-GFP), and 5(')NEP-GFP constructs lacking the NLS. 3(')NEP-GFP transfected cells showed plasma membrane/cytoplasmic fluorescence whereas the 5(')NEP-GFP fusion protein was also detected in the nucleus. The omission of the NLS resulted in no reduction in nuclear and an increase in cytoplasmic staining. The results suggest that the analyzed structural motifs determine the subcellular distribution of NEP in epithelial LNCaP PC cells and stromal prostatic cells and therefore could be responsible for the altered cellular localization of NEP observed in PC.