Mechanism of disordered subcellular localization of TFE3 fusion protein in TFE3 translocation renal cell carcinoma / 现代泌尿外科杂志

【Objective】 To investigate the effects of the loss of exon 1 of TFE3 on nuclear localization of chimeric TFE3 protein in TFE3 translocation renal cell carcinoma (TFE3 tRCC). 【Methods】 The localization of TFE3 protein in TFE3 tRCC and clear cell renal cell carcinoma (ccRCC) were detected with immunochemistry. The exon retention of TFE3 gene in TFE3 tRCC was analyzed in databases and literatures. The plasmids containing TFE3 full-length and different-length of TFE3 exons which were constructed to pCDH-MCS-EGFP-Puro were transfected into HEK293T using Lipo FiterTM. The localization of EGFP protein in HEK293T cells were detected with confocal microscopy. The localization of TFE3 protein and truncated TFE3 protein were detected with Western blotting. The mRNA expression of the downstream genes of TFE3 protein were detected with q-PCR. 【Results】 Strong nuclear signal of TFE3 protein was observed in TFE3 tRCC, whereas TFE3 protein in ccRCC was mainly localized in cytoplasm. The results of fluorescence imaging and Western blotting showed that TFE3 full-length protein was expressed both in nucleus and cytoplasm, and the expression of truncated TFE3 protein was mainly localized in nucleus. The q-PCR analysis demonstrated that the deletion of exon 1 in TFE3 gene led to a higher transcriptional level of targeted genes of TFE3 protein. 【Conclusion】 The loss of exon 1 in TFE3 played a critical role in preventing TFE3 protein from entering the nucleus. In TFE3 tRCC, the loss of exon 1 in TFE3 gene leads to the nuclear localization of TFE3 fusion protein and activation of its downstream target genes. This mechanism promises to uncover the occurrence and development of TFE3 tRCC.

The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects.

β-Actin facilitates etoposide-induced p53 nuclear import

As a tumor suppressor, p53 preserves genomic integrity in eukaryotes. However, limited evidence is availablefor the p53 shuttling between the cytoplasm and nucleus. Previous studies have shown that b-actin polymerization negatively regulates p53 nuclear import through its interaction with p53. In this study, we found thatDNA damage induces both b-actin and p53 accumulation in the nucleus. b-actin knockdown impaired thenuclear transport of p53. Additionally, b-actin could interact with p53 which was enhanced in response togenotoxic stress. Furthermore, N terminal deletion mutants of p53 shows reduced levels of association with bactin. We further identified Ser15, Thr18 and Ser20 of p53 are critical to the b-actin: p53 interaction, whichupon mutation into alanine abrogates the binding. Taken together, this study reveals that b-actin regulates thenuclear import of p53 through protein–protein interaction.

Molecular characterization and expression pattern analysis of a novel stress-responsive gene ‘BeSNAC1’ in Bambusa emeiensis

NAC transcription factors (TFs) are master regulators of environmental stresses exerting a crucial role in plant growth and development. However, the studies on NAC TFs from Bambusa emeiensis are scarce. In this investigation, a novel gene from B. emeiensis encoding NAC protein was cloned and characterized. The gene was isolated based on the amino acid sequence data of stress-responsive SNAC1 of rice, named ‘BeSNAC1 (accession no. MG763922)’. The full-length sequence of 1681 bp was found to contain an open-reading frame of 912 bp that encode a protein of 303 amino-acid residues. The multiple protein sequence alignments unveiled that BeSNAC1 contains a typical NAC domain. Additionally, the phylogenetic analysis showed that the corresponding protein belonged to the SNAC group, as it cladded with SNAC1, HvSNAC1, TaNAC2, SbSNAC1 and ZmSNAC1 proteins. Transactivation and subcellular localization assay disclosed that BeSNAC1 is a transcriptional activator localized in the cell nucleus.Moreover, the time-dependent expression pattern of BeSNAC1 was profiled under abscisic acid (ABA), polyethylene glycol 6000 (PEG-6000), NaCl, H2O2 and Na2SO4 treatments via a quantitative real-time polymerase chain reaction. The results revealed that the expression of BeSNAC1 was significantly upregulated in all treatments, a significant difference was observed under H2O2, NaCland ABA (P 0.001) and PEG and Na2SO4 (P < 0.01) treatments, respectively. Conclusively, our findings provide evidence that ‘BeSNAC1’ is a nuclear protein that might act as part of the transcription regulation complex and is involved in the ABA signalling pathway and abiotic stress tolerance mechanisms in B. emeiensis.

Titanium dioxide nanoparticles coupled with nuclear localization sequence increases the radiosensitivity of U251 glioma cells / 中华放射医学与防护杂志

Objective To investigate the effects of titanium dioxide ( TiO2 ) nanoparticles coupled with nuclear localization sequence ( NLS ) on the radiosensitivity of U251 glioma cells. Methods Synthesis and characterization of the TiO2-NLS nanoparticles with nuclear targeting property. U251 cells were treated with nanoparticles and/or ionizing radiation. Flow cytometry analysis was performed to measure the ROS content and the cell apoptotic percentage. The DNA damage was detected by using γ-H2AX foci staining. Clonogenic survival assay was used to evaluate the radiosensitivity of U251 cells. Results NLS modification promoted nuclear translocation of TiO2 nanoparticles. Compared with the control nanoparticles, TiO2-NLS treatment increased radiation-induced cell apoptosis (t=8. 96, P<0. 05). Clonogenic survival assay showed the radiosensitization ratios of TiO2 nanoparticle-treated group and TiO2-NLSnanoparticle-treated group were 1. 18 and was 1. 29, respectively. These two ratios had statistically difference ( t =14. 72, P< 0. 05). Conclusions Nuclear targeting TiO2 nanoparticles enhances the radiosensitivity of U251 glioma cells.

Experiment study on the transfection of exogenous genes promoted by ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal / 中华超声影像学杂志

Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery(UTMD) mediated a peptide nucleic acid (PNA) binding nuclear localization signal (NLS).Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor.The SV40T antigen receptors were expressed on the membranes of 293T cells.The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA,which increased nuclear localization.Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm,respectively.The study was divided into five groups:Contrast (group A),Common microbubble + DNA (group B),Antibody-targeted microbubbles + DNA (group C),Common microbubbles + NLS-PNA-DNA (group D),Antibody-targeted microbubbles + NLS-PNA-DNA (group E).Fluorescence microscope was used to observe the fluorescent light in each group;flow cytometry to test the transfection;RT-PCR and Western blot to detect genes' mRNA and protein expression level.Results Ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability(＞80％).Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully.The transfection results of flow cytometry were 0,(9.30 ± 0.46)％,(26.46 ± 2.01)％,(29.54 ± 0.62)％,(45.72 ± 1.86)％,respectively,and the differences were statistically significant(P ＜0.05).The relative mRNA and protein expression in group E were greater than those in group C and D respectively (P ＜0.05).Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nuclear DNA import.

Experimental study of combining ultrasound targeted microbubble destruction with nuclear localization signal peptide to enhance gene transfection in treatment of canines myocardial infarction / 中华超声影像学杂志

Objective To improve the canines myocardial infarction curative effect by using ultrasound targeted microbubble destruction (UTMD) combining with nuclear localization signal(NLS) peptide to increase hAng-1 gene transfection efficiency.Methods Forty-six canines were randomly divided into 4 groups (n=10 in each group) after the models of myocardial infarction were prepared.Group A were untreated control;Group B were transfected with hAng-1;Group C were transfected with UTMD+hAng-1;Group D were transfected with UTMD+NLS nuclear localization signal+hAng-1.The therapeutic agents were intravenously injected at one week after myocardial infarction in each group,and the ultrasound were irradiated at the precardium in group C and D.①Echocardiography was used before and at one week after myocardial infarction and 28 days after gene transfection.Two-dimensional echocardiography was used to detect cardiac function,the left ventricular ejection fraction,the left ventricular wall motion and the myocardial contrast echocardiography were used to detect myocardial perfusion of all canines in the four groups.②On twenty-eight days after gene transfection,mRNA and Western Blot were used to detect the expression of hAng-1.Immunohistochemistry was used to detect capillary density of peri-infarct area and microvessel density (MVD).Masson′s trichromatic staining and gross specimen were used to evaluate the degree and the area of myocardial fibrosis.The gene transfection efficiency and the curative effect of all the four groups were evaluated and compared.Results ①Before myocardial infarction,in four groups canine ventricular wall motion and cardiac function were normal,and myocardial filling defect was not showed by myocardial contrast echocardiography.At one week after myocardial infarction,the left ventricular anterior and interval walls motion and the left ventricular ejection fraction in the four groups were significantly decreased.Myocardial contrast echocardiography showed anterior and interval walls myocardial filling defect.There was no significant difference among the four groups(P>0.05);On 28 days after gene transfection the left ventricular ejection fraction in the four group were increased in an order of group A,B,C,D,there was significant difference when comparing group C and D with other groups separately(P<0.05).Myocardial contrast echocardiography showed much contrast filling in the infarction and surrounding area in group D,a little contrast filling in group C and filling defect in group A and group B,there was significant difference when comparing group C and D with other groups separately(P<0.05).②RT-PCR and Western Blot showed the hAng-1 mRNA and protein expression in group D were higher than those in the other group.There was significant difference when comparing group C and group D with other groups separately(P<0.05).③Immunofluorescence showed the capillary densities were(4.7±1.6)/mm2,(11.2±2.8)/mm2,(70.0±6.4)/mm2 and (85.3±7.0)/mm2 in group A,group B,group C and group D.The differences were statistically significant compared group C and group D with other groups (P<0.05).④Masson′s trichromatic staining and cardiac gross specimen showed that the degree and area of myocardial fibrosis were gradually reduced in an order of group A,B,C,D.Conclusions UTMD and NLS peptide could effectively transfect hAng-1 gene and it provided a novel strategy of gene treatment for ischemic heart disease.

The molecular mechanism for nuclear transport and its application / 대한해부학회지

Transportation between the cytoplasm and the nucleoplasm is critical for many physiological and pathophysiological processes including gene expression, signal transduction, and oncogenesis. So, the molecular mechanism for the transportation needs to be studied not only to understand cell physiological processes but also to develop new diagnostic and therapeutic targets. Recent progress in the research of the nuclear transportation (import and export) via nuclear pore complex and four important factors affecting nuclear transport (nucleoporins, Ran, karyopherins, and nuclear localization signals/nuclear export signals) will be discussed. Moreover, the clinical significance of nuclear transport and its application will be reviewed. This review will provide some critical insight for the molecular design of therapeutics which need to be targeted inside the nucleus.

The enhanced effect of nuclear localization signal peptide during the ultrasound targeted microbubbles ;destruction mediated gene transfection / 中华超声影像学杂志

Objective To investigate the transfection efficiency combining ultrasound targeted microbubbles destruction(UTMD)and nuclear localization signal(NLS)peptide for facilitating the plasmid of enhanced green fluorescent protein (pEGFP) into nucleus.Methods This study was divided into 3 groups,group A:UTMD+ pEGFP;group B:UTMD+ NLS + pEGFP;group C:Lipo3000 + pEGFP.The NLS was labeled by FITC and pEGFP was marked by Cy3.The different mole ratio was adjusted between NLS and pEGFP for observing the best ratio of combination.The human umbilical vein endothelial cells (HUVEC) were transfected by the optimum ultrasonic irradiation parameters and the optimal NLS∕pEGFP mole ratio.Six hours after transfection,the rate of Cy3 labeled pDNA into cells and nuclear were detected by flow cytometer and laser confocal microscope respectively.Forty-eight hours after transfection,the transfection efficiency was detected by flow cytometer;the survival rate of cells was measured by CCK8. RT-PCR and Western technology were used to detect the relative expression amount of mRNA and protein. The above indicators were compared among 3 groups,which were used to evaluate the enhanced effect of NLS in UTMD mediated gene transfection.Results ① Six hours after transfection,the NLS with green fluorescence and pEGFP with red fluorescence can show at the same site and signal intensity within the cell, that suggested a combination between them,agarose gel electrophoresis showed that the best molar ratio of NLS∕pEGFP combining was 10 4 ∶1 .②Six hours after transfection,the rates of pEGFP into the cells were (63±12)%,(80±10)% and(92±8)%;the rates of pEGFP into the nucleus were(1 7±3)%,(50±12)%and(35±8)% in 3 groups respectively(P 80% in all groups;the transfection efficiency,relative quantity of mRNA and protein expression were increased gradually.There were significant differences among 3 groups(P <0.05).They were 1 .6,2.3 and 2.4 times in group B than those in group A,still lower than those in group C.Conclusions The UTMD combining NLS can promote the pEGFP into nucleus for improving the transfection efficiency.The NLS peptide can play an enhanced effect as a new strategy of UTMD.

Nuclear expression of CXCR4 in renal cancer correlates with metastasis and poor survival / 第二军医大学学报

Objective To investigate the nuclear expression of CXC chemokine receptor 4 (CXCR4) in renal cancer, and analyze its relation with renal cancer metastasis and prognosis. Methods A total of 413 patients with renal cancer who were treated in our urologic center from Mar. 2011 to Nov. 2012 were included in the present study. The subcellular expression of CXCR4 was examined by immunofluorescence staining; the correlation between CXCR4 nuclear location and clinical features, prognosis was analyzed. Results We found that 170 of the 413 renal cancer patients were CXCR4 nuclear staining-positive (group A), and the rest 243 cases were CXCR4 nuclear staining-negative (group B); the two groups had matchable baseline data. Compared with group B, group A had significantly higher Robson stage (P <0.01), more frequent cancer embolus (P <0.01), more frequent lymphatic metastasis (P <0.01), and more frequent distant metastasis (P <0.01). The overall survival rate of group A (86.5%, 147/170) was significantly lower than that of group B (97.1%, 236/243; P <0.001). Conclusion Nuclear expression of CXCR4 in renal cancer tissues is associated with higher Robson stage, more frequent cancer embolus, more frequent lymphatic metastasis, more frequent distant metastasis and poor prognosis.

Location of NLS-RARα protein in peripheral blood tumor cells of patients with acute promyeolic leukemia / 第二军医大学学报

Objective To verify the presence and location of the NLS-RARex protein in the peripheral blood tumor cells of patients with acute promyeolic leukemia (APL). Methods Western blotting analysis was used to identify the NE enzyme in the peripheral blood tumor cells of APL patients. The nucleoprotein in tumor cells was prepared and NLS-RARex protein was detected by Western blotting analysis. The expression and location of NLS-RARex protein in peripheral blood tumor cells of APL patients were examined by FITC/DAPI double immunofluorescence staining and FITC/PI double staining laser confocal microscopy. The expression and location of NLS-RARex protein in NB4 cells infected with recombinant adenovirus Ad-NE was used as positive control and those of wildtype RARex in the neutrophils of healthy controls were taken as negative control. Results Positive control was successfully established. NE enzyme and NLS-RARex protein were expressed in peripheral blood tumor cells of APL patients. Immunofluorescence and laser confocal findings indicated that NL&RARex protein was mainly located at the nuclei of peripheral blood tumors cells in APL patients. Conclusion NLS-RARex protein has been successfully detected by 3 different methods in the peripheral blood tumor cells of APL patients and its intracellular location has also been proposed, which can contribute to the early diagnosis and recurrence monitoring of APL.

Screening of the nucleotide sequences affected by α-synuclein in vitro / 中国药理学通报

Aim To study the screening of the nucleo-tide sequences might be affected by α-syn in vitro. Methods The nucleotide sequences were synthesized according to the feature of base composition, and then mixed with the α-syn-GFP. The CD was used to ana-lyse the changes of the peak. Result The peak of the CD changed greatly when the α-syn-GFP mixed with the GC-box like sequence. Conclusion The α-syn-GFP might affect the GC-box like sequence after trans-located into the nuclei. Then, it plays a role in physio-logical and pathological conditions by affecting the reg-ulation of gene expression.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

Co-immunoprecipitation in screening proteins interacting with CXCR4 during nuclear localization in renal cell carcinoma A498 cells / 第二军医大学学报

Objective To screen for the proteins interacting with CXCR4 during nuclear localization in renal cell carcinoma (RCC) A498 cells. Methods Specific band in co-immunoprecipitation (Co-IP) experiments was sent for mass spectrometry. With the results of Co-IP experiments and mass spectrometry, the proteins interacting with CXCR4 were determined by bioinformatics analyses. Results Three specific bands were found after Co-IP with anti-CXCR4 antibody, and the results of mass spectrometry of the three specific bands showed 36 proteins possibly interacting with CXCR4. Bioinformatics analyses showed that NR1D2, c-src and HSPA8 might interact with CXCR4 and participate in CXCR4 nuclear localization. Conclusion NR1D2, c-src and HSPA8 might have participated in CXCR4 nuclear localization in RCC A498 cells.

The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-beta, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.

Ataxia telangiectasia mutated nuclear localization in head and neck cancer cells is PPP2R2B-dependent.

Background: Protein phosphatase 2A (PP2A) has been implicated in radiation-induced activation of cellular responses, likely by its ability to regulate the autophosphorylation of the ataxia telangiectasia mutated (ATM) protein, a key molecule involved in the DNA damage response initiated by double-stranded DNA breaks. Interestingly, a hereditary defect in the PPP2R2B gene, which encodes the beta isoform of PP2A regulatory subunit B, causes autosomal dominant spinocerebellar ataxia 12, a clinical condition resembling that of ataxia telangiectasia patients. Moreover, PPP2R2B is significantly downregulated in many human cancers, including head and neck squamous cell carcinomas (HNSCCs). Objective: Examine whether PPP2R2B regulates ATM function, thereby contributing to tumor progression due to the resulting defective DNA repair. Methods: The roles of PPP2R2B were evaluated in irradiated HNSCC cell lines, siRNAPPP2R2B cells and okadaic acid treated cells. Expression of PPP2R2B was measured by microarray, Western blot analysis and real time quantitative rtPCR. ATM quantity and localization, ATM phosphorylation and γ-H2AX were determined by Western blot analysis and/or immunofluorescence assay. Clonogenic cell survival assay was performed to determine ionizing radiation sensitivity. Results: PPP2R2B expression is reduced in multiple tumor types, including HNSCCs. Indeed, HNSCC cell lines that have lower PPP2R2B mRNA expression and siRNAPPP2R2B cells lower basal and radiation-induced levels of phosphorylated ATM and the consequent reduction in the levels of phosphorylation of the downstream ATM target, γ-H2AX. Depletion of PPP2R2B and inhibition of PP2A with okadaic acid resulted in limited ATM nuclear localization. Finally, siRNAPPP2R2B cells displayed enhanced sensitivity to death after radiation. Conclusion: In HNSCCs, ATM nuclear localization is PPP2R2B dependent, and decreased PPP2R2B expression may result in limited ATM activation by preventing its nuclear accumulation and ATM-chromatin interaction. Therefore, decreased PPP2R2B expression in HNSCCs may contribute to genomic instability, cancer development and radiation sensitivity by limiting ATM functions.

Preliminary study on CXCR4 nuclear localization sequence in metastatic renal cell carcinoma / 第二军医大学学报

Objective: To investigate the role of SDF-1/CXCR4 in metastasis of renal cell carcinoma and to observe the intracellular location of different CXCR4 segments in renal carcinoma cells. Methods: The potential nuclear localization sequences of different CXCR4 were discovered by nuclear localization software and experiments. Full length and truncated forms of CXCR4 were fused with green fluorescent protein pEGFP-N1 and their influence on subcellular localization was examined by confocal microscopy after transfecting them into renal carcinoma cell line A498. Results: Analysis with PSORT II Prediction revealed that the nuclear localization sequence region of CXCR4 was located between amino acids 146 and 149(RPRK). Expression products of the recombinant plasmids with SDF-1 stimulation, including EGFP-CXCR4 (1-510 bp), EGFP-CXCR4(1-765 bp) and wild-type EGFP-CXCR4, were mainly located in the cell nuclei. However,expression product of EGFP-CXCR4(1-267 bp) with SDF-1 stimulation was mainly located in the renal cell cytoplasm. Expression product of wild-type EGFP-CXCR4 full length plasmid without SDF-1 stimulation was mainly located in the cell cytoplasm; these results accorded with the results of bioinformatics analysis. Conclusion: Nuclear localization sequence of CXCR4 is located in the amino acids 90 to 170, which provides a theoretical basis for further clarifying the nuclear localization sequences of CXCR4 in renal cell carcinoma cells and for finding new potential target for inhibiting the metastasis of renal cell carcinoma.

Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells / 第二军医大学学报

Objective: To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method. Methods: The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells. Co-immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro. Results: Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody. Conclusion: The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.

The Nucleocytoplasmic Transport of Viral Proteins / 中国病毒学·英文版

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus / 中国病毒学·英文版

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.