生长抑制因子1基因核定位序列-绿荧光蛋白融合表达载体的构建及表达

目的构建p33^ING1b核定位序列（nuclear locating sequence，NLS）-绿荧光蛋白融合表达载体，将其转染到人胚肺纤维母细胞系MRC-5，建立稳定表达该融合蛋白的细胞模型。方法应用逆转录PCR获得p33^ING1b的NLS序列，然后将NLS序列插入绿荧光蛋白融合表达载体pEGFP-C1的多克隆位点，构建pEGFP-C1-NLS-绿荧光蛋白融合表达载体，再用此载体转染MRC-5细胞系，观察活细胞绿荧光蛋白的亚细胞定位。结果成功构建了pEGFP-C1-NLS-绿荧光蛋白融合表达载体，由该载体表达的绿荧光蛋白-NLS肽段融合蛋白产生的绿色荧光信号全部定位于胞核部位，而空载体转染的细胞表达的绿色荧光蛋白，绿色荧光信号定位于细胞浆中。结论在活细胞内，生理情况下p33^ING1b完全定位于细胞核，并且在其亚细胞定位的转运过程中，NLS肽段起着决定性作用。     

噬菌体显示技术在短肽库中的应用现状

噬菌体显示技术是近年来出现的一种新技术,它是将外源蛋白通过与丝状噬菌体外壳蛋白融合而将外源蛋白表达于噬菌体颗粒的表面。该技术已经被广泛地应用于噬菌体短肽库的构建。由于该表达的短肽可以与其相应的结合分子相识别而发挥其生物活性,因而,噬菌体短肽库技术在分子间识别机理的研究、蛋白工程的改造以及药物的筛选、疫苗的研制等方面具有广泛应用前景。本文综述了近年来噬菌体显示技术在短肽库中的应用进展。     

FXR基因家族

FXR基因家族是与脆性X综合征发病相关的一个基因家族。有三个家族成员。在进化上高度保守,氨基酸序列高度相似,有共同的功能结构域：两个KH结构域,RGG盒,NES和NLS。能够与RNA和多聚核蛋白体结合。通过NES和NLS携带其mRNA在细胞核和细胞质之间穿梭。不同的蛋白亚型其组织分布各异。该家族成员在大脑和神经发育过程中发挥重要作用,影响认知过程和智力发育。     

BDV核蛋白在少突胶质细胞内表达定位的基础研究

目的 研究博尔纳病病毒核蛋白在转染的少突胶质细胞(oligodendroglial cell,OL)内的表达定位及其对细胞活性的影响.方法 应用RT-PCR检测转染细胞内BDVp40的基因片段,应用激光共聚焦显微镜和Western blot的方法观察核蛋白在细胞内的表达定位.应用MTT方法观察核蛋白对OL活性的影响.结果 在转染BDVp40基因片段的荧光表达质粒的OL内检测到BDVp40的基因片段;激光共聚焦显微镜提示核蛋白存在于细胞质和细胞膜中;Western blot的结果显示在细胞膜蛋白中存在核蛋白,而在细胞质中未检测到该蛋白;MTT法检测BDV核蛋白对OL活性具有明显的抑制作用.结论 通过研究转染的OL内稳定表达了BDV核蛋白,并将其定位在细胞的结构蛋白中,尤其在细胞膜中含量更为丰富,说明其可能在细胞的信号转导或介导病毒感染人胞过程中发挥关键作用.BDV核蛋白对OL的活性有抑制作用,这可能是BDV导致中枢神经系统损伤的机制之一.     

α-突触核蛋白与帕金森病相互关系研究进展

α-突触核蛋白是一种可溶性小分子蛋白主要在中枢神经系统的突出前末梢表达。近年来关于α-突触核蛋白和帕金森病等神经退行性病的密切关系被广泛报道越来越多。在本综述中,详细介绍了α突触核蛋白的构成、生理功能和作用、病理生理、聚合机制等方面的研究进展,从α-突触核蛋白的角度理解帕金森病的发生机制。     

蛋白转导在基因治疗中的应用

蛋白转导是近几年生命科学领域发现的一种独特现象,具有蛋白转导功能的蛋白通过几个短的碱性小肽组成的蛋白转导域(proteintransductiondomain,PTD)的介导,能将与其共价连接的DNA、多肽或蛋白质以及其他大分子物质通过非经典途径穿过细胞膜甚至血脑屏障,并在细胞间自由传递。PTD这种能携带融合蛋白自由进入细胞的独特功能为人类一些疾病的基因治疗提供了一种新的运载工具,在基因治疗中有着美妙的应用前景。     

核定位信号在JC病毒样颗粒入核转运中的作用

目的探讨JC病毒(JCV)主要外壳蛋白VP1所含核定位信号(NLS)在JCV病毒样颗粒(VLP)入核转运中的作用。方法应用位点诱变法将JCV野生型VP1(wtVP1)氨基末端3个氨基酸:第5位赖氨酸、第6位精氨酸、第7位赖氨酸分别置换为丙氨酸、甘氨酸、丙氨酸,制备成NLS变异的VP1(ΔNLS-VP1)。编码ΔNLS-VP1的基因克隆到原核表达质粒pET-15b中,体外表达、重组VP1 NLS变异的病毒样颗粒(ΔNLS-VLP),异硫氰基荧光素(FITC)标记后感染地高辛渗透或未渗透的SVG细胞,荧光显微镜观察VLP入核转运。结果wtVP1组成的病毒样颗粒(wtVLP)可以进入地高辛渗透或未渗透的SVG细胞核,而ΔNLS-VLP只能进入细胞浆,不能进入细胞核;体外转染后,wtVP1主要在SVG细胞核表达,而ΔNLS-VP1主要在细胞浆表达;包裹外源性DNA的VLP感染SVG细胞后,则wtVLP包裹的DNA在细胞浆和细胞核内均可检测到,而ΔNLS-VLP包裹的DNA只能在细胞浆检测到,不能在细胞核内检测到。VLP体外结合分析发现,wtVLP与胞浆转运因子(importin)α、β均可结合,而ΔNLS-VLP与importinα、β均不能结合。结论VP1 NLS对于VLP入核转运是必需的,VIP的入核转运是由VP1 NLS与importin相互作用介导的。     

生长抑制蛋白4核定位信号和PHD指状结构域共同调控丝裂原活化蛋白激酶通路中ERK1的激活

目的探索生长抑制蛋白4(inhibitor of growth 4,ING4)的核定位信号(nuclear localization sig-nal,NLS)和PHD指状结构域(plant homeodomain)在刺激丝裂原活化蛋白激酶(MAPK)细胞信号通路成员ERK1磷酸化中的作用。方法通过构建ING4的NLS和PHD结构域突变或缺失重组质粒,将上述质粒分别与ERK1表达载体共转染HEK-293细胞,Western印迹检测ERK1总蛋白和磷酸化ERK1蛋白水平。结果在HEK-293细胞中,ING4蛋白分别缺失NLS和PHD结构域后,其对MAPK细胞信号通路成员ERK1磷酸化的促进作用均降低,突变ING4的NLS结构域中两个重要位点Lys-Lys(KK)和Arg-Ala-Arg-Ser-Lys(RARSK)并不改变对ERK1激活的能力,但突变ING4 PHD结构域中RKKK位点则改变了调控ERK1磷酸化的能力。结论ING4的NLS和PHD指状结构域同时参与了刺激MAPK细胞信号通路成员ERK1的激活。     

ERK信号通路的信号转导调控机制

胞外信号调控激酶（ERK）是发现的第1个丝裂原活化蛋白激酶（MAPK）,它调控多种重要的细胞生物学过程,包括细胞增殖、分化和凋亡等。ERK信号级联反应能够特异地介导广泛的生物学过程,其机制主要是通过信号的反馈调控,与支架蛋白的相互作用,亚细胞定位的改变,在级联反应的每一个环节存在不同功能的多种组分,细胞内非磷酸酶抑制物和G蛋白等的调控实现的。     

Karyopherins家族与核孔转运的研究进展

大分子物质入核是靠其核内定位序列 (NLS) ,而核内输出是靠其核输出信号 (NES)。不同的NLS和NES直接或靠配体间接的被转运受体识别。目前确定的转运受体都属于同一家族 -Karyopherins家族 ,它们可以在核和胞质间穿梭 ,可以与小的RanGTPase以及核孔蛋白相结合。RanGTPase调节转运受体与转运物、配体、核孔蛋白间的结合 ,而这是决定核孔转运的关键。然而一部分受体转运物复合物通过核孔复合体 (NPC)并不需要Ran水解GTP。     

Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1

Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.     

Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence

Background:

The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus.

Results:

We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coliβ-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells.

Conclusions:

The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.

     

Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10-13 and 41-42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-alpha, importin-beta nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41-72, which contained a nucleolar localization signal (NoLS) sequence. The 41-72 N peptide when fused to EGFP mimicked the nucleolar-cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus.     

Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C delta1

BACKGROUND: Although phospholipase C (PLC)delta1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCdelta1 contains an NLS sequence. RESULTS: A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCdelta1 in Madin-Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429-442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCdelta1 412-498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm. CONCLUSION: The nuclear import of PLCdelta1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.     

Structure-function relationship of the nuclear localization signal sequence of parathyroid hormone-related protein

Parathyroid hormone-related protein (PTHrP) contains a nuclear localization signal (NLS) sequence within 87-107. NLS sequences are generally capable of penetrating cellular membranes due to a richness of basic amino acid residues, and thus have been used as cell-penetrating peptides (CPPs) to translocate biologically active peptides/proteins into cells. The NLS sequence of PTHrP is not exception to this finding; however, PTHrP(87-107) contains 2 acidic glutamate residues at 99 and 101 within the basic amino acid stretch, which is not commonly observed in other CPPs such as HIV-1 Tat(48-60). In this study, we indicated structure-function relationship of the PTHrP NLS to understand the effect of acidic glutamate residues on cell permeability and intracellular localization. We chemically synthesized PTHrP(87-107) and its N-terminally truncated analogues. Their intracellular localization pattern was analyzed by microscopy, radioimmunoassay, and fluorescence-activated cell sorting. Although all analogues were translocated into cells, internalization by the cytoplasm and/or nucleus was length-dependent; specifically, PTHrP(97-107), PTHrP(95-107), and PTHrP(93-107) were more frequently localized in the cytoplasm. We assume that reduction in the net positive charge within PTHrP NLS analogues resulted in increased cytoplasm- translocation activity. We propose that PTHrP(97-107) is a useful carrier peptide for delivery and expression of cargo molecules in the cytoplasm.     

Differential properties of two putative nuclear localization sequences found in the carboxyl-terminus of human ifn-gamma.

Interferon-gamma (IFN-gamma), a protein that uses the Jak-Stat pathway for signal transduction, translocates rapidly to the nucleus in cells treated extracellularly with the cytokine. A nuclear localization sequence (NLS) has been identified and characterized in the C-terminus of IFN-gamma. Both human and murine IFN-gamma contain this NLS. We show in this report that human IFN-gamma (HuIFN-gamma) contains a second NLS at an upstream site, as determined in standard import assays using digitonin-permeabilized HeLa cells. The primary sequence, analogous with the NLS sequence identified in murine IFN-gamma, representing amino acids 122-132 of HuIFN-gamma was capable of mediating the nuclear import of the autofluorescent protein allophycocyanin (APC) in an energy-dependent manner. The second sequence, representing amino acids 78-92 of HuIFN-gamma, was also capable of mediating the nuclear import of APC in an energy-dependent manner but to a greatly reduced extent. The nuclear import of both sequences conjugated to APC was strongly blocked by competition with unconjugated HuIFN-gamma(122-132). Competition by the sequence HuIFN-gamma(78-92) effectively blocked the import of APC-conjugated HuIFN-gamma(78-92) but, at the same concentration, was not capable of inhibiting the nuclear import of APC-conjugated HuIFN-gamma(122-132), suggesting that HuIFN-gamma(78-92) was a less efficient NLS than HuIFN-gamma(122-132). This is consistent with >90% loss of antiviral activity of HuIFN-gamma lacking the downstream NLS in 122-132. The nuclear import of APC-conjugated HuIFN-gamma(122-132) was inhibited by a peptide containing the prototypical polybasic NLS of the SV40 T NLS, which suggests that the same Ran/importin cellular machinery is used in both cases.     

A smart polymeric platform for multistage nucleus-targeted anticancer drug delivery

Tumor cell nucleus-targeted delivery of antitumor agents is of great interest in cancer therapy, since the nucleus is one of the most frequent targets of drug action. Here we report a smart polymeric conjugate platform, which utilizes stimulus-responsive strategies to achieve multistage nuclear drug delivery upon systemic administration. The conjugates composed of a backbone based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer and detachable nucleus transport sub-units that sensitive to lysosomal enzyme. The sub-units possess a biforked structure with one end conjugated with the model drug, H1 peptide, and the other end conjugated with a novel pH-responsive targeting peptide (R8NLS) that combining the strength of cell penetrating peptide and nuclear localization sequence. The conjugates exhibited prolonged circulation time and excellent tumor homing ability. And the activation of R8NLS in acidic tumor microenvironment facilitated tissue penetration and cellular internalization. Once internalized into the cell, the sub-units were unleashed for nuclear transport through nuclear pore complex. The unique features resulted in 50-fold increase of nuclear drug accumulation relative to the original polymer–drug conjugates in vitro, and excellent in vivo nuclear drug delivery efficiency. Our report provides a strategy in systemic nuclear drug delivery by combining the microenvironment-responsive structure and detachable sub-units.