双组分核定位信号介导Apoptin定位于肿瘤细胞核

Apoptin是一种来源于鸡贫血病毒的小蛋白,在肿瘤细胞中定位于细胞核,而在正常细胞中主要分布于细胞质。根据预测,Apoptin分子中有2段序列(NLS1和NLS2)可能是单组分核定位信号。通过基因突变和缺失构建了Apoptin各种不同的核定位信号突变体和磷酸化突变体,利用增强型绿色荧光蛋白(EGFP)作标签,观察了其在肿瘤细胞中亚细胞定位的变化。结果表明,NLS1和NLS2单独均不是有效的单组分核定位信号。Apoptin的核定位信号是由NLS1和NLS2这2段序列共同组成的双组分核定位信号,缺少任何一段序列都会严重影响Apoptin在肿瘤细胞中的核定位。其中,NLS2对于Apoptin的核定位起主要作用。Apoptin的获得型磷酸化突变体并不能转位到正常细胞的细胞核中,而其磷酸化负突变体仍定位于肿瘤细胞的细胞核。另外,丝氨酸／苏氨酸蛋白激酶抑制剂H7也不影响Apoptin在肿瘤细胞中的核定位。很可能,Apoptin的磷酸化并不参与调控其核定位信号的功能。     

真核表达载体pEGFP—N1-VP3的构建及在SGC-7901细胞中的表达和定位

构建真核表达载体pEGFP-N1-VP3并稳定转染人胃癌细胞SGC-7901,观察EGFP-VP3融合蛋白在肿瘤细胞中的分布和亚细胞定位,探讨凋亡素诱导肿瘤细胞凋亡的机制.用PCR技术扩增出(凋亡素)VP3基因片段,克隆至载体pEGFP-N1,鉴定无误后,将构建的重组质粒pEGFP-N1-VP3经脂质体介导转染SGC-7901细胞,在荧光显微镜和激光扫描共聚焦显微镜下观察凋亡素在肿瘤细胞中的分布、亚细胞定位.用AO/EB荧光染色法检测其在体外诱导肿瘤细胞凋亡的效应.经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入载体pEGFP-N1,稳定转染细胞中EGFP-VP3在肿瘤细胞中得以高表达,转染后逐渐从细胞质迁移至细胞核,最后定位于细胞核内.AO/EB荧光染色观察到大量细胞凋亡.结论:成功构建真核表达载体pEGFP-N1-VP3,并成功培养出表达绿色荧光蛋白和凋亡素的SGC-7901稳定细胞株.EGFP-VP3融合蛋白在肿瘤细胞中具有核定位效应,并诱导肿瘤细胞凋亡.     

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.     

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     

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。     

鸡贫血病毒VP3蛋白序列与其核定位功能的相关性

鸡贫血病毒（chicken anemia virus, CAV）编码一种小蛋白VP3.通过构建vp3基因与绿色荧光蛋白基因的真核融合表达载体,转染5种肿瘤/转化细胞株, 观察绿色荧光在亚细胞区域的定位,证实VP3具有核定位的功能;分析VP3的氨基酸序列,将其具核定位序列（nuclear localization sequence, NLS）特征的区域删除,再构建此缺失的VP3的基因与绿色荧光蛋白基因的真核融合表达载体、转染实验显示核定位现象消失;进一步将具核定位序列特征的区域亚克隆到绿色荧光蛋白真核表达载体上,核定位功能再现.从而推断这一区域是VP3蛋白核定位的功能区.此区域位于VP3的C端,且富含碱性氨基酸,二级结构预测发现这一区域形成特定的β折叠结构.用碘丙锭(propidium iodide, PI)染色的方法还得到了VP3促进肿瘤细胞凋亡的初步证据.     

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.     

STAT3 入核分子机制的初步研究

为了研究Stat3入核的分子机制,将SV40大T 抗原的经典核定位序列NLS(nuclear localization sequence)分别融合在Stat3-GFP分子和缺失突变体Dstat3-GFP的分子之间,构建Stat3-NLS-GFP和Dstat3-NLS-GFP融合分子。转染293T细胞,以NLS-GFP为阳性对照,通过激光共聚焦显微镜的观察融合分子的亚细胞位置,未经白介素-6刺激的Stat3-NLS-GFP和经白介素-6刺激的Stat3-GFP呈胞核分布,未经白介素-6刺激的Stat3-GFP和Dstat3-NLS-GFP呈胞浆分布. 初步证明Stat3入核是由于获得了核定位序列。     

BRD7核定位信号的结构分析和功能鉴定

目的:对BRD7的核定位信号进行预测、结构分析和功能鉴定,并考察其对BRD7亚细胞定位的影响。方法:通过生物信息学对BRD7的核定位信号进行预测和结构分析,然后利用绿色荧光蛋白(GFP)介导的直接荧光和间接免疫荧光定位方法分别对核定位信号的功能进行鉴定,并考察其对BRD7亚细胞定位的影响。结果:BRD7的65～96位氨基酸残基具有潜在核定位信号(NLS)的结构特征,该核定位信号包含3簇碱性氨基酸残基,可视为由2个紧密相邻、部分重叠的双向核靶序列NLS1和NLS2组成;并发现NLS及其构成上的NLS1和NLS2均具有介导异源蛋白GFP胞核定位的功能,从而证实BRD7的65～96位残基为BRD7功能性核定位信号所在区域,且单簇碱性氨基酸残基的缺失不足以破坏其核定位信号的功能;同时发现野生型BRD7呈胞核分布,而核定位信号缺失型BRD7主要呈胞浆分布。结论:BRD7的65～96位氨基酸残基为BRD7功能性核定位信号所在区域,在BRD7胞核分布模式中发挥了十分重要的作用。     

STAT3 入核分子机制的初步研究

为了研究Stat3入核的分子机制,将SV40大T抗原的经典核定位序列NLS(nuclear localization sequence)分别融合在Stat3-GFP分子和缺失突变体Dstat3-GFP的分子之间,构建Stat3-NLS-GFP和Dstat3-NLS-GFP融合分子。转染293T细胞,以NLS-GFP为阳性对照,通过激光共聚焦显微镜的观察融合分子的亚细胞位置,未经白介素-6刺激的Stat3-NLS-GFP和经白介素-6刺激的Stat3-GFP呈胞核分布,未经白介素-6刺激的Stat3-GFP和Dstat3-NLS-GFP呈胞浆分布,初步证明Stat3入核是由于获得了核定位序列。     

核糖体蛋白L6/Taxreb107的核定位信号的分析

核糖体蛋白L6（RpL6,Taxreb107）含有三个具有核定位信号特征的基序.用作者构建的核定位信号捕获系统分析了这些核定位信号是否具有介导蛋白质进行核转位的功能.将RpL6/Taxreb107分段插入核定位信号捕获载体的克隆位点后转化宿主酵母,发现其前两个核定位信号可以介导融合蛋白进入细胞核,而第三个核定位信号无此作用.将RpL6/Taxreb107分段与绿色荧光蛋白融合后转染培养的哺乳类细胞,证实了以上在酵母中所得的结果.进一步发现RpL6/Taxreb107的前两个核定位信号同时具有核仁定位的功能.当在细胞中表达的早期,进入核内的融合蛋白优先定位于核仁.这些结果一方面有助于理解RpL6/Taxreb107核转位的机理,同时说明作者构建的核定位信号捕获系统也可用在蛋白质中寻找核定位信号.     

HPV16L1核浆运输的动力学过程

利用增强型绿色荧光蛋白(Enhancegreenflurenscentprotein,EGFP)标记不同的截短型HPV16L1蛋白(Humanpapillomavirustype16L1protein,HPV16L1),分析HPV16L1蛋白核定位信号(Nucleuslocationsignal,NLS)的作用。构建重组pFB-EGFP、pFB-EGFP-HPV16L1、pFB-EGFP-HPV16L1△NLS和pFB-EGFP-NLSHPV16L1p转移载体;在DH10Bac宿主菌内经Tn7转座子介导的同源重组后转染Sf-9细胞,获得重组Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS和Ac-EGFP-NLSHPV16L1杆状病毒,感染Sf-9昆虫细胞表达相应截短型HPV16L1融合蛋白;利用荧光显微镜和激光共聚焦显微镜观察不同融合蛋白的荧光特性和核浆转运动力学过程。结果发现Ac-EGFP杆状病毒感染的Sf-9细胞内明亮的绿色荧光均匀分布;重组Ac-EGFP-HPV16L1和Ac-EGFP-NLSHPV16L1杆状病毒感染的Sf-9细胞,明亮的绿色荧光主要位于细胞核内;重组Ac-EGFP-HPV16L1△NLS杆状病毒感染的Sf-9细胞,绿色荧光局限于细胞浆内,细胞核内无绿色荧光。说明HPV16L1蛋白羧基端的23个氨基酸(GKRKATPTTSSTSTTAKRKKRKL)具有完全核定位作用,能引导HPV16L1蛋白和EGFP突破核膜屏障进入Sf-9细胞核内。     

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.     

Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.     

Tus,an E. coli Protein,Contains Mammalian Nuclear Targeting and Exporting Signals

Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.     

A switching system regulating subcellular localization of nuclear proteins using a viral protease

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (nbeta-gal), within the cytoplasm, nbeta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nbeta-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nbeta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.     

介导BLM解旋酶细胞核定位的关键氨基酸位点鉴定

BLM解旋酶是人RecQ DNA解旋酶家族重要成员之一,在机体的DNA复制、重组、损伤修复以及维护基因组稳定性等方面发挥重要作用。早期研究表明,BLM解旋酶通过自身携带的核定位信号（nuclear localization signal, NLS）进入细胞核,但是介导其细胞核定位的关键氨基酸位点尚不清楚。本研究构建了BLM解旋酶C端(aa642 1417)截短体克隆,首先通过截短表达的方法确证其NLS结构域。在此基础上,构建重组真核表达载体pEGFP NLS/BLM NES/Rev,通过观察BLM NLS碱性氨基酸位点突变对EGFP NLS/ BLM NES/Rev融合蛋白细胞核定位的影响,以此快速鉴定NLS中介导BLM解旋酶细胞核定位的关键氨基酸位点。结果表明,BLM(aa642 1417) C端截短体具有与全长BLM解旋酶相同的细胞核定位,同时确证1344RSKRRK1349是BLM解旋酶NLS结构域的活性位点,且具有与SV40 NLS相同的核输入能力。氨基酸位点突变试验结果表明,R1344A、K1346A、R1348A和K1349A点突变均减少了EGFP NLS/BLM NES/Rev和EGFP BLM(642 1417)融合蛋白的细胞核定位。因此,这4个位点是介导BLM解旋酶细胞核定位的关键氨基酸位点。此结果为后续研究BLM解旋酶细胞核定位的分子机制奠定了基础。     

Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Nuclear protein transport processes have largely been studied using in vitro semi‐intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T‐ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T‐ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM‐1‐mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells. J. Cell. Biochem. 107: 1160–1167, 2009. © 2009 Wiley‐Liss, Inc.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.