Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit

Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II. Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells. Our results indicated that EGFP-EcCdtB-II localized to the nucleus. The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants. When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II. These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II.     

Dynamics and assembly of the cytolethal distending toxin

The cytolethal distending toxin (CDT) is a widespread bacterial toxin that consists of an active subunit CdtB with nuclease activity and two ricin-like lectin domains, CdtA and CdtC, that are involved in the delivery of CdtB into the host cell. The three subunits form a tripartite complex that is required to achieve the fully active holotoxin. In the present study we investigate the assembly and dynamic properties of the CDT holotoxin using molecular dynamics simulations and binding free energy calculations. The results have revealed that CdtB likely adopts a different conformation in the unbound state with a closed DNA binding site. The two characterized structural elements of the aromatic patch and groove on the CdtA and CdtC protein surfaces exhibit high mobility, and free energy calculations show that the heterodimeric complex CdtA-CdtC, as well as the CdtA-CdtB and CdtB-CdtC sub-complexes are less energetically stable as compared to the binding in the tripartite complex. Analysis of the dynamical cross-correlation map reveals information on the correlated motions and long-range interplay among the CDT subunits associated with complex formation. Finally, the estimated binding free energies of subunit interactions are presented, together with the free energy decomposition to determine the contributions of residues for both binding partners, providing insight into the protein-protein interactions in the CDT holotoxin.     

Differences in crystal and solution structures of the cytolethal distending toxin B subunit: Relevance to nuclear translocation and functional activation

Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233-Asp242). Residues Leu186-Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined alpha-helix and beta-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions.     

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.     

Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity

Acinetobacter baumannii is an emerging opportunistic pathogen responsible for healthcare-associated infections. The outer membrane protein A of A. baumannii (AbOmpA) is the most abundant surface protein that has been associated with the apoptosis of epithelial cells through mitochondrial targeting. The nuclear translocation of AbOmpA and the subsequent pathology on host cells were further investigated. AbOmpA directly binds to eukaryotic cells. AbOmpA translocates to the nucleus by a novel monopartite nuclear localization signal (NLS). The introduction of rAbOmpA into the cells or a transient expression of AbOmpA–EGFP causes the nuclear localization of these proteins, while the fusion proteins of AbOmpAΔNLS–EGFP and AbOmpA with substitutions in residues lysine to alanine in the NLS sequences represent an exclusively cytoplasmic distribution. The nuclear translocation of AbOmpA induces cell death in vitro . Furthermore, the microinjection of rAbOmpA into the nucleus of Xenopus laevis embryos fails to develop normal embryogenesis, thus leading to embryonic death. We propose a novel pathogenic mechanism of A. baumannii regarding the nuclear targeting of the bacterial structural protein AbOmpA.     

细胞致死性膨胀毒素损伤宿主防御机制研究进展

细胞致死性膨胀毒素(cytolethal distending toxin, CDT)属于AB₂毒素,由多种革兰氏阴性菌产生。CDT是第一种被描述的细菌基因毒素,编码3种多肽：CDTA、CDTB和CDTC。CdtB是活性部分,有损伤多种细胞类型的能力。CDT具有一种新的分子作用模式,会干扰真核细胞周期的进展,从而导致G2/M停滞和细胞凋亡,该作用机制针对细胞,而且现阶段对于CDT的研究更多也是细胞层面,但是CDT作为毒力因子最终作用是损伤宿主造成疾病。但目前对CDT与宿主相互作用的分子机制了解尚不清晰。本文对细胞致死性膨胀毒素作为毒力因子从损伤上皮屏障、适应性免疫以及促进炎症反应三方面来综合阐述其对宿主防御机制途径的损伤,以期了解其致病机制以及为其临床治疗提供理论依据和新思路。     

Requirement of phosphatidylinositol 3-kinase activity for translocation of exogenous aFGF to the cytosol and nucleus

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.     

Characterization of Putative Cholesterol Recognition/Interaction Amino Acid Consensus-Like Motif of Campylobacter jejuni Cytolethal Distending Toxin C

Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.     

CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.     

Synthetic peptides containing a region of SV40 large T-antigen involved in nuclear localization direct the transport of proteins into the nucleus

We studied the mechanism of transport of proteins into the nucleus using synthetic peptides containing the nuclear location signal sequence of Simian virus 40 (SV 40) large T-antigen. When chick erythrocytes containing a synthetic large T-antigen nuclear translocation signal were fused with SV 40-transformed human fibroblasts, the migration of native large T-antigen into the chick nuclei was suppressed. Migration of proteins detected by human specific antinuclear autoimmune antibody was not blocked. An analog of the nuclear location signal peptide did not inhibit entry of large T-antigen into the chick nuclei. This result suggests that the peptide blocked the migration of only native large T-antigen into the nucleus, and that the signal of the majority of nuclear proteins for nuclear transport is not the same as that of the large T-antigen. The synthetic peptide was conjugated chemically with bovine serum albumin (BSA) and introduced into the cytoplasm of cultured human cells by red blood cell ghost-mediated microinjection. The BSA-synthetic peptide conjugate was found predominantly in the nucleus within 2 h after its introduction into the cells. BSA conjugated with the cross-linking reagent alone was not transported into the nucleus. Acetylated synthetic peptide was not effective in promoting nuclear localization of BSA. Mild trypsin treatment of the BSA-synthetic peptide conjugate suppressed nuclear localization. Conjugates of the synthetic peptide with phycoerythrin (Mr about 150 kD) and with secretory IgA (Mr about 380 kD) were both found in the nucleus very shortly after their introduction into the cytoplasm. These results suggest that the synthetic peptide containing the nuclear location signal sequence provides exogenous proteins with the ability to migrate into the nucleus. But, since a conjugate of the synthetic peptide with IgM (Mr about 940 kD) did not migrate into the nucleus after its microinjection, there may be a size limit in nuclear transport of conjugated proteins.     

Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions

The coevolution of bacterial pathogens and their hosts has contributed to the development of very complex and sophisticated functional pathogen--host interfaces. Thus, well-adapted pathogens have evolved a variety of strategies to manipulate host cell functions precisely. For example, a group of unrelated Gram-negative pathogenic bacteria have evolved a toxin, known as cytolethal distending toxin (CDT), that has the ability to control cell cycle progression in eukaryotic cells. Recent studies have identified CdtB as the active subunit of the CDT holotoxin. Through its nuclease activity, CdtB causes limited DNA damage, thereby triggering the DNA-damage response that ultimately results in the observed arrest of the cell cycle. In addition, it has been established that CDT is a tripartite AB toxin in which CdtB is the active 'A' subunit and CdtA and CdtC constitute the heterodimeric 'B' subunit required for the delivery of CdtB into the target cell. The mechanism of action of CDT suggests that the infliction of limited damage could be a strategy used by pathogenic bacteria to modulate host cell functions.     

Functional studies of the recombinant subunits of a cytolethal distending holotoxin

Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA , cdtB and cdtC . Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans , we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid-based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.     

Nuclear localization of the testis determining gene product SRY

We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family.     

核定位信号及其在病毒感染机制中的研究进展

核定位信号介导的蛋白入核是细胞内信号传递网络中核内外物质信息交流的重要一环,绝大多数病毒蛋白进入细胞核均需要核质转运受体识别和结合入核蛋白携带的核定位信号序列。病毒蛋白的入核转运机制在病毒感染过程中起着至关重要的作用,对于病毒的复制、毒力具有重要意义,针对该机制的研究有利于新的抗病毒靶点的发现。本文对核定位信号的分类信息进行了总结,并对不同的核质转运受体及其介导的入核机制进行了比较分析,概述了病毒入核蛋白及其核定位信号在病毒感染机制中的研究发现。     

Nuclear targeting determinants of the phage P1 cre DNA recombinase

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for genomic manipulation in mice and other eukaryotes. Because Cre is of prokaryotic origin, the 38 kDa protein has been presumed to gain access to the eukaryotic nucleus simply because it is sufficiently small to pass through the nuclear pore by passive diffusion. Instead, we show here that Cre carries nuclear targeting determinants that efficiently direct Cre entry into the nucleus of mammalian cells. Fusions of Cre with green fluorescent protein (GFP) identified two regions that are necessary for nuclear localization. Region I contains a cluster of basic amino acids that is essential for nuclear localization and which resembles a bipartite-like nuclear localization signal. Region II exhibits a beta-sheet structure with which the bipartite motif may interact. However, neither region is by itself sufficient for nuclear localization. Nuclear transport in vitro with a 98 kDa GFP-Cre fusion protein shows that Cre does not gain access to the nucleus by passive diffusion, but instead enters the nucleus by means of an energy-dependent process. Thus, Cre is one of the few prokaryotic proteins that have been shown to carry determinants that allow it to target the eukaryotic nucleus.     

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.