Nuclear translocation and DNAse I-like enzymatic activity of Acinetobacter baumannii outer membrane protein A

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a potential virulence factor that induces host cell death. Based on previous findings that AbOmpA translocated into the nuclei of host cells, the cell-death mechanism of AbOmpA through the nuclear targeting was investigated. Acinetobacter baumannii secreted AbOmpA in in vitro culture. The recombinant AbOmpA (rAbOmpA) was internalized by the host cells. The intracellular rAbOmpA was degraded into several forms of subfragments in the cytosol and then two subfragments of rAbOmpA translocated into the nuclei. The rAbOmpA exhibited the divalent cation-dependent endonuclease activity. In an in vivo assay with microinjection of rAbOmpA into the nucleus of fertilized Xenopus laevis eggs, rAbOmpA degraded chromosomal DNA with the characteristic DNA ladders and induced degeneration of the embryos. These results suggest that AbOmpA translocates into the nuclei of host cells and degrades chromosomal DNA by DNAse I-like enzymatic activity, which is a new pathogenic strategy of A. baumannii.     

Global gene expression patterns and induction of innate immune response in human laryngeal epithelial cells in response to Acinetobacter baumannii outer membrane protein A

The outer membrane protein A of Acinetobacter baumannii (AbOmpA) is an important pathogen-associated molecular pattern that induces host cell death. We determined the gene expression profiles of human laryngeal epithelial HEp-2 cells in response to the sublethal concentration of recombinant AbOmpA (rAbOmpA) and investigated the molecular mechanisms by which rAbOmpA induces an innate immune response. The microarray analysis showed that rAbOmpA sequentially regulated a relatively small set of genes, including those associated with signal transductions and molecules involved in immune response. Among the differentially expressed genes involved in innate immune responses, the surface expression of Toll-like receptor 2 and the production of inducible nitric oxide synthase (iNOS) were prominently observed. However, rAbOmpA did not induce the production of proinflammatory cytokines and chemokines. rAbOmpA activated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs). Inhibition of JNK MAPK suppressed iNOS production in the rAbOmpA-treated HEp-2 cells. These results suggest that interaction of laryngeal epithelial cells with AbOmpA has a significant impact on the induction of innate immunity during the early stages of A. baumannii infection.     

A novel nuclear localization signal in human DNA topoisomerase I

DNA topoisomerase (topo) I is a nuclear enzyme that plays an important role in DNA metabolism. Based on conserved nuclear targeting sequences, four classic nuclear localization signals (NLSs) have been proposed at the N terminus of human topo I, but studies with yeast have suggested that only one of them (amino acids (aa) 150-156) is sufficient to direct the enzyme to the nucleus. In this study, we expressed human topo I fused to enhanced green fluorescent protein (EGFP) in mammalian cells and demonstrated that whereas aa 150-156 are sufficient for nuclear localization, the nucleolar localization requires aa 157-199. More importantly, we identified a novel NLS within aa 117-146. In contrast to the classic NLSs that are rich in basic amino acids, the novel NLS identified in this study is rich in acidic amino acids. Furthermore, this novel NLS alone is sufficient to direct not only EGFP into the nucleus but also topo I; and the EGFP.topo I fusion driven by the novel NLS is as active in vivo as the wild-type topo I in response to the topo I inhibitor topotecan. Together, our results suggest that human topo I carries two independent NLSs that have opposite amino acid compositions.     

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.     

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.     

Protein transduction of an antioxidant enzyme: subcellular localization of superoxide dismutase fusion protein in cells

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.     

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.     

A non-canonical transferable signal mediates nuclear import of simian immunodeficiency virus Vpx protein

Protein transport into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly evident that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Vpx, a 112 amino acid protein from human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency virus (SIV) is one such protein, which does not have an identifiable canonical NLS and is yet efficiently imported to the nuclear compartment. Here we report that Vpx protein is imported to the nucleus independently of virus-encoded cofactors. When fusions of truncated versions of Vpx with full-length beta-galactosidase (beta-Gal) were tested, the region from Vpx 61 to 80 was found to be sufficient to mediate the import of the heterologous cytoplasmic protein to the nucleus. Inactivation of Vpx NLS precluded nuclear import of Vpx and reduced virus replication in non-dividing macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral nuclear import were present. Importantly, we identified and characterized a novel type of 20 amino acid transferable nuclear import signal in Vpx that is distinct from other import signals described. In addition, we show that the minimal nuclear targeting domain identified here overlaps with helical domain III (amino acid (aa) 64-82) and the structural integrity of this helical motif is critical for the nuclear import of Vpx. Taken together, these data suggest that Vpx is imported to the nucleus via a novel import pathway that is dependent on its 20 amino acid unique nuclear targeting signal, and that the nuclear import property of Vpx is critical for the optimal virus replication in non-dividing cells such as macrophages.     

A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II

Primary sequences of proteins often contain motifs that serve as "signatures" for subcellular targeting, such as a nuclear localization signal (NLS). However, many nuclear proteins do not harbor a recognizable NLS, and the pathways that mediate their nuclear translocation are unknown. This work focuses on CRABP-II, a cytosolic protein that moves to the nucleus upon binding of retinoic acid. While CRABP-II does not contain an NLS in its primary sequence, such a motif could be recognized in the protein's tertiary structure. We map the retinoic acid-induced structural rearrangements that result in the presence of this NLS in holo- but not apo-CRABP-II. The signal, whose three-dimensional configuration aligns strikingly well with a "classical" NLS, mediates ligand-induced association of CRABP-II with importin alpha and is critical for nuclear localization of the protein. The ligand-controlled NLS "switch" of CRABP-II may represent a general mechanism for posttranslational regulation of the subcellular distribution of a protein.     

Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.     

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 yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional- lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.     

Nuclear translocation of proteins and the effect of phosphatidic acid

Transport of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors called importins, typically dimmers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complexes (NPCs). However, how proteins without canonical NLS move into the nucleus is not well understood. Recent results indicate that phospholipids, such as phosphatidic acid, play important roles in the intracellular translocation of proteins between the nucleus and cytoplasm.     

Determinants of the nuclear localization of the heterodimeric DNA fragmentation factor (ICAD/CAD)

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.     

A photoregulated ligand for the nuclear import receptor karyopherin alpha.

The ability to orchestrate the transport of proteins between nucleus and cytoplasm provides cells with a powerful regulatory mechanism. Selective translocation between these compartments is often used to propagate cellular signals, and it is an intimate part of the processes that control cell division, viral replication, and other cellular events. Therefore, precise experimental control over protein localization, through the agency of light, would provide a powerful tool for the study and manipulation of these events. To this end, a prototype photoregulated nuclear localization signal (NLS) was derived from a native NLS. A library of 30 mutants of the bipartite NLS from Xenopus laevis nucleoplasmin containing a novel, photoisomerizable amino acid was prepared by parallel, solid-phase synthesis and screened in vitro for binding to the nuclear import receptor karyopherin alpha, which mediates the nuclear import of cellular proteins. A single peptide was identified in which the cis and trans photoisomers bind the receptor differentially. The strategy used to obtain this peptide is systematic and empirical; therefore, it is potentially applicable to any peptide-receptor system.     

Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Glucose-regulated GRP58 has shown clinical applications to endoplasmic reticulum (ER) stress and cancer. GRP58 is localized in the cytosol, endoplasmic reticulum (ER) and nucleus. Twenty-four amino acids at the N-terminal hydrophobic region are known to target GRP58 to ER for synthesis at the ER membrane and translocation into the ER lumen. In addition, GRP58 contains putative nuclear localization (494KPKKKKK500) and ER retention (502QEDL505) signals. However, the role of these signals in nuclear import and ER retention of GRP58 remains unknown. Present studies investigated the signals that control nuclear localization and ER retention of GRP58. Deletion/mutation of nuclear localization signal (NLS) abrogated nuclear import of GRP58. NLS attached to EGFP localized EGFP in the nucleus. However, deletion/mutation of putative ER retention signal alone did not alter ER retention of GRP58. Interestingly, a combined deletion/mutation of NLS and ER retention signals blocked the GRP58 retention in the ER. These results concluded that overlapping NLS and ER retention signal sequences regulate nuclear localization and ER retention of GRP58.     

A lily pollen ASR protein localizes to both cytoplasm and nuclei requiring a nuclear localization signal

LLA23, an abscisic acid-, stress- and ripening-induced (ASR) protein, was isolated previously from lily ( Lilium longiflorum ) pollen. Close examination of the C-terminus of this ASR protein revealed the presence of basic regions reminiscent of a nuclear localization signal (NLS). Fluorescence microscopy studies using green fluorescent protein (GFP) fusion proteins indicated that the bipartite NLS in LLA23 exhibited nuclear localization properties. Accordingly, mutations in the NLS motifs of LLA23 defined two regions, either of which was necessary for partial nuclear targeting and both of which were required for complete nuclear localization. In addition, oligonucleotide-directed mutagenesis identified lysine residues within the NLS necessary for nuclear localization. Immunogold localization confirmed that the protein was located to both the cytoplasm and nucleus of generative and vegetative cells of pollen grains; the generative nuclei showed the highest number of LLA23 labelling. The possible function of ASR proteins in both the cytoplasm and nuclei of pollen grains is discussed.     

Ran-unassisted Nuclear Migration of a 97-kD Component of Nuclear Pore–targeting Complex

A 97-kD component of nuclear pore-targeting complex (the β-subunit of nuclear pore–targeting complex [PTAC]/importin/karyopherin) mediates the import of nuclear localization signal (NLS)-containing proteins by anchoring the NLS receptor protein (the α-subunit of PTAC/importin/karyopherin) to the nuclear pore complex (NPC). The import requires a small GTPase Ran, which interacts directly with the β-subunit. The present study describes an examination of the behavior of the β-subunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically injected β-subunit rapidly migrates into the nucleus. The use of deletion mutants reveals that nuclear migration of the β-subunit requires neither Ran- nor α-subunit–binding but only the NPC-binding domain of this molecule, which is also involved in NLS-mediated import. Furthermore, unlike NLS-mediated import, a dominant-negative Ran, defective in GTP-hydrolysis, did not inhibit nuclear migration of the β-subunit. In the digitonin-permeabilized cell-free import assay, the β-subunit transits rapidly through the NPC into the nucleus in a saturating manner in the absence of exogenous addition of soluble factors. These results show that the β-subunit undergoes translocation at the NPC in a Ran-unassisted manner when it does not carry α-subunit/NLS substrate. Therefore, a requirement for Ran arises only when the β-subunit undergoes a translocation reaction together with the α-subunit/NLS substrate. The results provide an insight to the yet unsolved question regarding the mechanism by which proteins are directionally transported through the NPC, and the role of Ran in this process.