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。     

核因子κB的跨核膜转运及其调控机制

核因子kappaB(NF-κB)是一组重要的转录调节因子,当细胞处于静息状态时,它与抑制蛋白IκB结合以非活性的形式存在于胞浆中.当细胞受到多种外界信号刺激,NF-κB、IκB分别在核定位信号（NLS）的介导下经核孔复合物（NPC）转运入核.在核内,NF-κB与IκB再次结合成复合物,在核转出信号（NES）介导下,经CRM1依赖的途迳出核.该过程是能量依赖的主动转运过程,涉及小分子Ran蛋白及多种可溶性因子.     

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.     

八肋游仆虫第二类肽链释放因子核定位信号分析

蛋白质合成终止过程中肽链释放因子负责终止密码子的识别.真核生物第二类肽链释放因子（eRF3）是一类GTP酶,协助第一类肽链释放因子（eRF1）识别终止密码子和水解肽酰 tRNA酯键.之前的研究表明,两类肽链释放因子在细胞核中发挥功能,参与蛋白质合成和纺锤体的组装.本研究根据软件预测结果,构建了一系列八肋游仆虫eRF3的截短型突变体,分析在其N端是否存在引导eRF3的核定位信号.结果表明,在eRF3的N端有两个区域（NLS1:23-36 aa 和 NLS2: 236-272 aa）可以引导eRF3进入细胞核中,而且这两个区域具有典型的核定位信号的氨基酸序列特征. eRF3的核定位与其作为一种穿梭蛋白的功能相一致,即参与细胞有丝分裂纺锤体的形成和无义介导的mRNA降解途径.     

原型泡沫病毒Bel1蛋白核定位信号精细定位及相互作用核输入蛋白初探

Bel1是原型泡沫病毒(Prototype foamy virus,PFV)的反式激活因子,在病毒的复制周期中发挥关键作用。研究表明,Bel1含有核定位信号(Nuclear localization signal,NLS),但其精确氨基酸组成尚不明确,介导其入核的核输入蛋白亦未见报道。本研究利用插入Bel1截短片段的EGFP-GST融合表达体系,通过绿色荧光观察其亚细胞定位,首次精确确定Bel1NLS序列为215PRQKRPR221;定点突变明确了K218、R219和R221为Bel1核定位的必需残基,证明Bel1NLS属单分型核定位信号;GST-Pulldown实验显示这段序列可与α核输入蛋白(Importinα/Karyopherin alpha,official symbol:KPNA)KPNA1、KPNA6和KPNA7相互作用,即Bel1可能藉此转运入核。     

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。     

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.     

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。     

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.     

伪狂犬病毒UL49基因核定位信号的初步确定

伪狂犬病毒UL49基因编码的蛋白VP22是一种皮层蛋白,其生物学功能尚不清楚。预测并确定PRV UL49的核定位信号,可为研究其编码蛋白VP22的蛋白转导功能及作用机理奠定基础。通过生物信息学软件分析预测到,PRV UL49基因存在潜在的两个核定位信号:5~21位氨基酸序列(RKTRVA ADETASGARRR)NLS1和241~247位氨基酸序列(PGRKGKV)NLS2。本文将实验分为对照组、NLS1和NLS2同时缺失组、NLS1缺失组和NLS2缺失组四组,并通过PCR扩增、分子克隆等技术获得PRV Ea株UL49基因。以pEYFP-N1为载体,将UL49基因及各组缺失或突变片段插入EYFP上游,成功构建PRV pUL49-EYFP重组质粒及一系列缺失突变体,转染COS-7细胞后观察荧光定位。实验结果表明,确认预测到的NLS1和NLS2具有核输入功能,且位于241-247位的氨基酸序列(PGRKGKV)的入核功能更强,5~21位氨基酸序列(RKTRVA ADETASGARRR)为双向核定位信号,其同时具有核输出的功能。     

Induction of apoptosis by NORE1A in a manner dependent on its nuclear export

The RASSF family proteins were identified as tumor suppressors in a variety of human cancers, and evidenced distinct subcellular localization patterns among their subfamilies and isoforms. In this study, we showed that NORE1A was exported actively via its nuclear export signal (NES) in the C-terminus (residues 372-379). Substitutions of three lysine residues of NORE1A NES to alanines (L372, 376, 379A) showed its localization to the dot structures of the nucleus, which was similar to the NORE1A localizations observed after the administration to cells of Leptomycin B, a nuclear export inhibitor. The NORE1A NES mutant inhibited caspase-mediated apoptosis, whereas wild-type NORE1A induced caspase-3 activation. Furthermore, the NORE1A NES mutant did not co-localize with GFP-MST1, the direct downstream target of NORE1A. These results show that the nuclear export of NORE1A via NES is involved in the NORE1A-mediated induction of apoptosis.     

Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s)

Caspase-3 is thought to play an important role(s) in the nuclear morphological changes that occur in apoptotic cells and many nuclear substrates for caspase-3 have been identified despite the cytoplasmic localization of procaspase-3. Therefore, whether activated caspase-3 is localized in the nuclei and how active caspase-3 has access to its nuclear targets are important and unresolved questions. Here we confirmed nuclear localizations for both caspase-3-p17 and caspase-3-p12 subunits of active caspase in apoptotic cells using subcellular fractionation analysis. We also prepared polyclonal and monoclonal antibodies specific for active caspase-3 to define the subcellular localization of active caspase-3. Immunocytochemical observations using anti-active caspase-3 antibodies showed nuclear accumulation of active caspase-3 during apoptosis. In addition, caspase-3, but not caspase-7, translocated from the cytoplasm into the nucleus after induction of apoptosis. Mutations at the cleavage site between the p17 and p12 subunits and the substrate recognition site for the P3 amino acid of the DXXD substrate cleavage motif inhibited nuclear translocation of caspase-3, indicating that nuclear transport of active caspase-3 required proteolytic activation and substrate recognition. These results suggest that active caspase-3 is translocated in association with a substrate-like protein(s) from the cytoplasm into the nucleus during progression through apoptosis.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Identification of a nuclear localization signal and importin beta members mediating NUAK1 nuclear import inhibited by oxidative stress

NUAK1 is a serine/threonine kinase member of the AMPK-α family. NUAK1 regulates several processes in tumorigenesis; however, its regulation and molecular targets are still poorly understood. Bioinformatics analysis predicted that the majority of NUAK1 localizes in the nucleus. However, there are no studies about the regulation of NUAK1 subcellular distribution. Here, we analyzed NUAK1 localization in several human cell lines, mouse embryo fibroblasts, and normal mouse tissues. We found that NUAK1 is located in the nucleus and also in the cytoplasm. Through bioinformatics analysis and studies comparing subcellular localization of wild type and NUAK1 mutants, we identified a conserved bipartite nuclear localization signal at the N-terminal domain of NUAK1. Based on mass spectrometry analysis, we found that NUAK1 interacts with importin-β members including importin-β1 (KPNB1), importin-7 (IPO7), and importin-9 (IPO9). We confirmed that importin-β members are responsible for NUAK1 nuclear import through the inhibition of importin-β by Importazole and the knockdown of either IPO7 or IPO9. In addition, we found that oxidative stress induces NUAK1 cytoplasmic accumulation, indicating that oxidative stress affects NUAK1 nuclear transport. Thus, our study is the first evidence of an active nuclear transport mechanism regulating NUAK1 subcellular localization. These data will lead to investigations of the molecular targets of NUAK1 according to its subcellular distribution, which could be new biomarkers or targets for cancer therapies.     

Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.     

A putative NES mediates cytoplasmic localization of Apoptin in normal cells

Chicken anemia virus (CAV) is a small non-envelopedvirus containing a single-stranded circular DNA genome.The virus causes a disease in the newborn chickens, whichis characterized by generalized lymphoid atrophy, increasedmortality and severe anemia. CAV …     

Comprehensive studies on subcellular localizations and cell death-inducing activities of eight GFP-tagged apoptosis-related caspases

By using green fluorescent protein fusion, we investigated the subcellular localization of all the caspases that have been cloned from humans and implicated in the execution of apoptosis. We divided these caspases into three groups according to subcellular localization. The first group includes caspase-1, -3, -6, -7, and -9, which are expressed mainly in the cytoplasm with various levels of nuclear localization depending on the cell type. The second group has a single member, caspase-2, which is primarily localized in the nucleus. The nuclear localization was demonstrated to be mediated by a nuclear localization signal near the NH(2)-terminus of the prodomain. The third group includes caspase-8 and -10, which have a cytoplasmic distribution. These two members have potent, rapid cell death-inducing activity and are prone to make aggregates when overexpressed. Their prodomains formed marked fibrous structures in the cytoplasm whose localization seemed distinct from organelles or cytoskeletons. None of the GFP-caspases examined in this study showed a predominant mitochondrial localization as has been reported for some caspases.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.