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.     

Molecular characterization of nucleus-localized RNA-binding proteins from higher plants

We report the isolation and characterization of genes from the higher plants Arabidopsis, spinach and tobacco which code for nucleus-localized RNA-binding proteins. Common features of these polypeptides are glycine/arginine-rich regions with several RGG repeats at their N- and C-termini, which are sufficient for RNA binding in northwestern assays. All polypeptides analysed contain two basic bipartite nuclear localization signals and translational fusions harbouring these regions with the -glucuronidase gene direct the fusion proteins into the nucleus. Nuclear localization was confirmed by cellular fractionation with a polyclonal antiserum raised against the over-expressed tobacco protein NtRGG1p. Two or three copies of related RGG genes appear to be present in the analysed organisms and the expression of some of them is regulated: a tobacco gene is light-regulated and a spinach gene is preferentially expressed in roots. Possible biological functions of this class of RNA-binding proteins as well as structure/function relationships related to the modular structure are discussed.     

Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita

The total numbers of nuclei in giant cells induced by Meloidogyne incognita in pea, lettuce, tomato, and broad bean were determined. Mature giant cells from pea had the most nuclei per giant cell with a mean of 59 ± 23, lettuce had the fewest with 26 ± 16, and tomato and broad bean were intermediate. The rate of increase in numbers of nuclei for all plant species was greatest during the first 7 days after inoculation. No mitotic activity was observed in giant cells associated with adult nematodes. Number of nuclei per giant cell doubled each day during the period of greatest mitotic activity, but number of total chromosomes per giant cell increased 20-fold per day at the same time. The hypothesis is presented that factor(s) responsible for the polyploid, mulfinucleate condition characteristic of giant cells may be different from factor(s) responsible for aneuploid numbers of chromosome per nucleus or for nuclear aberrations such as the presence of linked nuclei.     

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。     

Importins and Beyond: Non-Conventional Nuclear Transport Mechanisms

The movement of proteins between the cytoplasm and the nucleus conventionally involves the recognition of nuclear targeting signals by members of the importin (Imp) superfamily of nuclear transporters, followed by translocation through the nuclear envelope-embedded nuclear pore complexes (NPCs). It is becoming increasingly apparent, however, that distinct alternative pathways for nuclear transport exist and are relatively abundant. This review examines several of these novel pathways, including facilitation of Imp-dependent transport by microtubule motors, and Imp-independent pathways involving either other transport molecules such as the calcium-binding protein calmodulin or through direct binding to the components of the NPC. The existence of these pathways and the fact that many proteins appear to possess separate Imp-dependent and -independent nuclear import mechanisms ensure that the cell can function under conditions in which Imp-dependent transport is inhibited and/or modulate the efficiency of Imp-dependent transport itself, according to the need.     

The effect of protein supplied in the growth medium on plant pathogen resistance

Externally supplied protein (bovine serum albumin, BSA) affects root development of Arabidopsis, increasing root biomass, root hair length, and root thickness. While these changes in root morphology may enhance access to soil microenvironments rich in organic matter, we show here that the presence of protein in the growth medium increases the plant''s resilience to the root pathogen Cylindrocladium sp.     

Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich nuclear targeting signals

Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. In addition to the known role in tumorigensis, the activated form of Ras exhibits growth-inhibitory effects by unknown mechanisms. Several Ras effector proteins identified as mediators of apoptosis and cell-cycle arrest also exhibit properties normally associated with tumor suppressor proteins. Here, we show that Ras effector RASSF5/NORE-1 binds strongly to K-Ras but weakly to both N-Ras and H-Ras. RASSF5 was found to localize both in the nucleus and the nucleolus in contrast to other Ras effector proteins, RASSF1C and RASSF2, which are localized in the nucleus and excluded from nucleolus. A 50 amino acid residue transferable arginine-rich nucleolar localization signal (NoLS) identified in RASSF5 is capable of interacting with importin-beta and transporting the cargo into the nucleolus. Surprisingly, similar arginine-rich signals identified in RASSF1C and RASSF2 interact with importin-alpha and transport the heterologous cytoplasmic proteins to the nucleus. Interestingly, mutation of arginine residues within these nuclear targeting signals prevented interaction of Ras effector proteins with respective transport receptors and abolished their nuclear translocation. These results provide evidence for the first time that arginine-rich signals are able to recognize different nuclear import receptors and transport the RASSF proteins into distinct sub-cellular compartments. In addition, our data suggest that the nuclear localization of RASSF5 is critical for its cell growth control activity. Together, these data suggest that the transport of Ras effector superfamily proteins into the nucleus/nucleolus may play a vital role in modulating Ras-mediated cell proliferation during tumorigenesis.     

Axo-glial interactions regulate the localization of axonal paranodal proteins

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain- bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin beta/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.     

Nuclear and cytoplasmic localization of neural stem cell microRNAs

Although generally regarded as functional in the cytoplasm, a number of microRNAs (miRNAs) have been found in the nucleus, possibly with a role in gene regulation. Here we report that, in fact, a substantial fraction of all human miRNAs are present in the nucleus of neural stem cells. Further, subsets of these miRNAs display consistently higher standardized rank in the nucleus than in the cytoplasm of these cells, as identified with an RT-qPCR technology and confirmed by microarray analysis. Likewise, other miRNAs display higher cytoplasmic standardized ranks. Three samples were partitioned into nuclear and cytoplasmic fractions in six assays for 373 miRNAs. From the 100 most highly expressed miRNAs, standard scores of nuclear and cytoplasmic concentrations were determined. Among those, 21 miRNAs had all three nuclear standard scores higher than all three cytoplasmic scores; likewise, 31 miRNAs had consistently higher cytoplasmic scores. Random concentrations would result in only five in each set. Remarkably, if one miRNA has a high standard score in a compartment, then other miRNAs having the same 5' seeds and certain similar 3' end patterns are also highly scored in the same way. That is, in addition to the seed sequence, 3' sequence similarity criteria identify families of mature miRNAs with consistently high nuclear or cytoplasmic expression.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Escherichia coli tmRNA lacking pseudoknot 1 tags truncated proteins in vivo and in vitro

Transfer-messenger RNA (tmRNA) and protein SmpB facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3′ end of mRNA templates lacking stop codons. The tmRNA molecule is a hybrid of tRNA- and mRNA-like domains that are usually connected by four pseudoknots (pk1–pk4). Replacement of pk1 with a single-stranded RNA yields pk1L, a mutant tmRNA that tags truncated proteins very poorly in vitro but very efficiently in vivo. However, deletion of the whole pk1 is deleterious for protein tagging. In contrast, deletion of helix 4 yields Δh4, a fully functional tmRNA derivative containing a single hairpin instead of pk1. Further deletions in the pk1 segment yield two subclasses of mutant tmRNAs that are unable to tag truncated proteins, but some of them bind to stalled ribosomes. Our studies demonstrate that pk1 is not essential for tmRNA functions but contributes to the stability of the tmRNA structure. Our studies also indicate that the length of this RNA segment is critical for both tmRNA binding to the ribosome and resumption of translation.     

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.     

Effect of hypochlorous acid solution on the eradication and prevention of Pseudomonas aeruginosa infection,serum biochemical variables,and cecum microbiota in rats

In this study, hypochlorous acid solution, a weak acid, provided as drinking water to rats, was evaluated for its ability to eradicate and prevent Pseudomonas aeruginosa infection, while monitoring its simultaneous effect on serum biochemical variables and microbiota in the rat cecum. The results suggest that the solution could not eliminate the bacteria in the experimentally infected rats; however, the administration of a 10-parts-per-million (ppm) hypochlorous acid solution as drinking water was effective in inhibiting horizontal spread of P. aeruginosa infection among cage mates. Additionally, exposure to hypochlorous solution did not have any effect on serum biochemical variables of the rat including levels of total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total bilirubin, lipase, amylase, urea nitrogen, total protein, calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), except for potassium (K) levels. The most frequently isolated bacteria in the rat cecum included species belonging to Bacteroidales, Lactobacillus, Clostridiales, Erysipelotrichaceae, Akkermansia, Coriobacteriales, and Firmicutes. The ratio of the terminal restriction fragment length polymorphism (T-RFLP) peaks did not differ across rats administered with 5 and 10 ppm weak acid solution as compared to the control group for any of the bacteria, except for Erysipelotrichaceae and Firmicutes, where the ratio of T-RFLP peaks was higher in the 5 ppm group for Erysipelotrichaceae and in the 10 ppm group for Firmicutes than that in the control group (P<0.01). The results suggest that the weak acid hypochlorous solution could not eradicate P. aeruginosa completely from rats. The solution was effective in preventing infection without affecting serum biochemical variables; however, some of bacterial microbiota may have changed due to administration of the solution.     

Centromere Protein B Null Mice are Mitotically and Meiotically Normal but Have Lower Body and Testis Weights

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.     

Production and Partial Characterization of Stylet Exudate from Adult Females of Meloidogyne incognita

Adult females of Meloidogyne incognita were excised from tomato roots and incubated in 0.04 M phosphate buffered saline, pH 7.4 for 18-72 hours to allow accumulation of stylet exudate. Twenty-four percent of the females produced exudate during the initial 18-hour incubation period; 70% of those females producing exudate initially produced additional exudate during the subsequent 54-hour incubation period. Analysis of exudate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least nine major protein bands. Differential staining with silver and Coomassie Brilliant Blue G-250 stains indicated that three of the bands were glycoproteins. Upon acid hydrolysis, 14 amino acids were detected in the stylet exudate. The basic amino acids lysine, histidine, and arginine comprised 21.8% of the total amino acids detected. No peroxidase activity was detected in the stylet exudates. Data presented extend and generally confirm prior work on the chemical composition of stylet exudate.     

On the translocation of proteins across the chloroplast envelope

Most of the chloroplast proteins are coded for in the nucleus and are synthesized in the cytosol from where they are subsequently transported into the different chloroplast compartments. The structural properties of the N-terminal extensions (transit peptides) of these nuclear-coded precursor proteins are discussed as well as the energy requirements for their translocation and the involvement of receptor proteins and that of other (ATP-dependent) factors.     

鸭源新城疫病毒M蛋白核定位信号突变影响病毒的毒力和复制能力

【目的】研究鸭源新城疫病毒(Newcastle disease virus,NDV)M蛋白核定位信号(nuclear localization signal,NLS)突变对其毒力和复制能力的影响。【方法】利用鸭源NDV SS1株P基因和F基因上的AgeⅠ和Bstz17Ⅰ酶切位点,将overlapPCR方法获得的M蛋白NLS突变的片段替换到p NDV/SS1GFP中获得全长质粒pNDV/SS1GFP-M/NLSm。通过反向遗传学技术拯救M蛋白NLS突变体病毒,并对拯救的病毒进行血凝(hemagglutination,HA)试验、荧光试验和M基因测序鉴定。另外,对突变体病毒进行M蛋白的亚细胞定位观察,以及病毒的生物学特性、空斑形成能力和体外增殖能力测定。【结果】成功构建M蛋白NLS突变的全长质粒pNDV/SS1GFP-M/NLSm。细胞转染物接种鸡胚后的第1代尿囊液无HA效价,盲传3代才能检测到拯救病毒的HA效价。进一步的荧光试验和M基因测序确定拯救的病毒是突变体病毒r SS1GFP-M/NLSm。与亲本病毒rSS1GFP相比,突变体病毒M蛋白由细胞核定位变为细胞质定位。此外,突变体病毒的毒力、在鸡胚上的复制能力以及在细胞中的空斑形成能力显著降低,并且感染细胞后产生的细胞病变轻微,M蛋白和绿色荧光蛋白的表达量均降低,说明M蛋白NLS突变使病毒的体外增殖能力受到抑制。【结论】NLS突变导致的M蛋白细胞核定位功能丧失可明显降低鸭源NDV的毒力和复制能力。