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D A Jans

Publications and source records attributed to D A Jans.

At least 19 recordsLinked to original sources
PubMed2006

Apoptin is modified by SUMO conjugation and targeted to promyelocytic leukemia protein nuclear bodies.

Apoptin, a protein of the chicken anemia virus (CAV), represents a novel potential anticancer therapeutic, because it induces apoptotic death specifically in tumor but not normal cells. The cellular localization appears to be crucial for apoptin's selective toxicity. In normal cells apoptin remains in the cytoplasm, whereas in transformed cells it migrates into the nucleus and kills the cell. However, the manner by which apoptin is able to distinguish between tumor and normal cells is unknown. Here, we report for the first time that apoptin interacts directly with the promyelocytic leukemia protein (PML) in tumor cells and accumulates in PML nuclear bodies (NBs), which are involved in apoptosis induction and viral replication. We also demonstrate that apoptin is sumoylated and that a sumoylation-deficient apoptin mutant is no longer recruited to PML-NBs, but localizes in the nuclear matrix. This mutant fails to bind PML, but can still induce apoptosis as efficiently as wild-type apoptin. Moreover, apoptin kills also PML-/- cells and promyelocytic leukemia cells with defective PML expression. Our results therefore suggest that apoptin kills tumor cells independently of PML and sumoylation, however, the interaction of apoptin with PML and small ubiquitin-like modifier (SUMO) proteins might be relevant for CAV replication.

Capsid Proteins↗
PubMed2006

Quantitative analysis of localization and nuclear aggregate formation induced by GFP-lamin A mutant proteins in living HeLa cells.

Although A-type lamins are ubiquitously expressed, their role in the tissue-specificity of human laminopathies remains enigmatic. In this study, we generate a series of transfection constructs encoding missense lamin A mutant proteins fused to green fluorescent protein and investigate their subnuclear localization using quantitative live cell imaging. The mutant constructs used included the laminopathy-inducing lamin A rod domain mutants N195K, E358K, M371K, R386K, the tail domain mutants G465D, R482L, and R527P, and the Hutchinson-Gilford progeria syndrome-causing deletion mutant, progerin (LaA delta50). All mutant derivatives induced nuclear aggregates, except for progerin, which caused a more lobulated phenotype of the nucleus. Quantitative analysis revealed that the frequency of nuclear aggregate formation was significantly higher (two to four times) for the mutants compared to the wild type, although the level of lamin fusion proteins within nuclear aggregates was not. The distribution of endogenous A-type lamins was altered by overexpression of the lamin A mutants, coexpression experiments revealing that aberrant localization of the N195K and R386K mutants had no effect on the subnuclear distribution of histones H2A or H2B, or on nuclear accumulation of H2A overexpressed as a DsRed2 fusion protein. The GFP-lamin fusion protein-expressing constructs will have important applications in the future, enabling live cell imaging of nuclear processes involving lamins and how this may relate to the pathogenesis of laminopathies.

Active Transport, Cell Nucleus↗
PubMed2005

Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates.

Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.

Cell Nucleus↗
PubMed2005

Drivers of germ cell maturation.

Spermatogenesis requires progression of germ line stem cells through a precisely ordered differentiation pathway to form spermatozoa. Diverse and dynamic signals from the transforming growth factor-beta (TGF-beta) superfamily influence many stages of germ cell development. For example, interactions between several TGF-beta superfamily ligands (bone morphogenetic proteins, activin, and glial-derived neurotrophic growth factor [GDNF]) appear to govern the onset of spermatogenesis, and we are exploring how germ cells interpret these competing signals. We examined the in vivo impact of activin on testis development using two mouse models, the inhba-/- mouse (which lacks the gene encoding the activin A subunit and dies at birth) and BK mice, with inhbb (encoding the activin betaB subunit) replacing inhba (which survive to adulthood and show delayed fertility onset in males). Distinct effects on Sertoli cell and germ cell populations during fetal and early postnatal development were measured. We recognize that specific proteins, including downstream targets of TGF-beta signals, such as Smads, must move into the nucleus to implement the gene transcription changes required for development. We hypothesized that changes at the level of cellular nuclear transport machinery may be required to mediate this. Examination of proteins involved in classical nuclear import, the importins, revealed that each importin has a developmentally regulated expression pattern in male germ cells. Because each importin binds a selected range of cargo proteins and mediates their nucleocytoplasmic passage, our findings suggest that each importin ferries cargo required for discrete stages of spermatogenesis.

Activins↗
PubMed2003

Nuclear import of the pre-integration complex (PIC): the Achilles heel of HIV?

Current treatments against the Aquired immune deficiency syndrome (AIDS) are reasonably effective in reducing the amount of human immunodeficiency virus (HIV) present in infected patients, but their side-effects, and the emergence of drug-resistant HIV strains have intensified the renewed search for novel anti-HIV therapies. An essential step in HIV infection is the integration of the viral genome into the host cell chromosomes within the nucleus. Unlike other retroviruses, HIV can transport its genetic material, in the form of the large nucleoprotein pre-integration complex (PIC), into the nucleus through the intact nuclear envelope (NE). This enables HIV to infect non-dividing cells such as macrophages and microglial cells. Detailed knowledge of the signal-dependent pathways by which cellular proteins and RNAs cross the NE has accumulated in the past decade, but although several different components of the PIC have been implicated in its nuclear import, the mechanism of nuclear entry remains unclear. Since specifically inhibiting PIC nuclear import would undoubtedly block HIV infection in non-dividing cells, this critical step of HIV replication is of great interest as a drug target. This review examines the complex and controversial literature regarding three PIC components--the HIV proteins matrix, integrase and Vpr--proposed to facilitate PIC nuclear import, and existing models of HIV PIC nuclear import. It also suggests approaches to move towards a better understanding of PIC nuclear import, through examining the role of individual PIC components in the context of the intact PIC by direct visualisation, in order to develop new anti-HIV therapeutics.

Acquired Immunodeficiency Syndrome↗
PubMed2001

The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1.

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.

Amino Acid Sequence↗
PubMed2001

Biophysical characterization of interactions involving importin-alpha during nuclear import.

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K(D) = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K(D) = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K(D) = 1.7 x 10(-8) m; nucleoplasmin NLS, K(D) = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.

Active Transport, Cell Nucleus↗
PubMed2001

Novel properties of the nucleolar targeting signal of human angiogenin.

The polypeptide ligand angiogenin, a potent inducer of angiogenesis, localizes in the nucleus/nucleolus subsequent to endocytosis by relevant cell types. This study examines the kinetic properties of the nucleolar targeting signal (NTS) of angiogenin (IMRRRGL(35)) at the single cell level. We show that the NTS is sufficient to target green fluorescent protein (GFP), but not beta-galactosidase, to the nucleolus of rat hepatoma cells. Mutation of Arg(33) to Ala within the NTS abolishes targeting activity. Nuclear/nucleolar import conferred by the NTS of angiogenin is reduced by cytosolic factors as well as ATP and is independent of importins and Ran. The NTS also confers the ability to bind to nuclear/nucleolar components which is inhibited by ATP hydrolysis; nonhydrolysable GTP analogs prevent nuclear accumulation in the absence of an intact nuclear envelope through an apparent cytoplasmic retention mechanism. Since the lectin wheat germ agglutinin does not inhibit transport, we postulate a mechanism for angiogenin nuclear/nucleolar import involving passive diffusion of angiogenin through the nuclear pore and NTS-mediated nuclear/nucleolar retention, and with cytoplasmic retention modulating the process. This pathway is clearly distinct from that of conventional signal-mediated nuclear protein import.

Active Transport, Cell Nucleus↗
PubMed2001

Nuclear import of Creb and AP-1 transcription factors requires importin-beta 1 and Ran but is independent of importin-alpha.

Although the specific role of transcription factors (TFs) is nuclear, surprisingly little is known in quantitative terms regarding the pathways by which TFs localize in the nucleus. In this study, we use direct binding assays, native gel electrophoresis, and fluorescence polarization measurements to show for the first time that the cAMP-response element binding protein (CREB) and related AP-1 and jun and fos constituents are recognized by importin beta1 (Impbeta) with nanomolar affinity. We reconstitute the nuclear import of these TFs in vitro, demonstrating dependence on cytosolic factors, and show that this is due to the requirement for Impbeta, since antibodies to Impbeta, but not to importin alpha (Impalpha), inhibit nuclear accumulation significantly. We show that Impbeta is necessary and sufficient for docking of CREB at the nuclear envelope; that Ran is essential for CREB nuclear import is demonstrated by the reduction of nuclear accumulation effected by RanGTPgammaS but not RanGDP, and by dissociation of the Impbeta-CREB-GFP complex by RanGTPgammaS but not RanGDP as demonstrated using fluorescence polarization assays. The results support the existence of an Impbeta1- and Ran-mediated nuclear import pathway for CREB and related constitutively nuclear TFs, which is Impalpha-independent and thus distinct from import pathways utilized by inducible TFs.

Active Transport, Cell Nucleus↗
PubMed2001

Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation.

Human mutations in the transcription factor SOX9 cause campomelic dysplasia/autosomal sex reversal. Here we identify and characterize two novel heterozygous mutations, F154L and A158T, that substitute conserved "hydrophobic core" amino acids of the high mobility group domain at positions thought to stabilize SOX9 conformation. Circular dichroism studies indicated that both mutations disrupt alpha-helicity within their high mobility group domain, whereas tertiary structure is essentially maintained as judged by fluorescence spectroscopy. In cultured cells, strictly nuclear localization was observed for wild type SOX9 and the F154L mutant; however, the A158T mutant showed a 2-fold reduction in nuclear import efficiency. Importin-beta was demonstrated to be the nuclear transport receptor recognized by SOX9, with both mutant proteins binding importin-beta with wild type affinity. Whereas DNA bending was unaffected, DNA binding was drastically reduced in both mutants (to 5% of wild type activity in F154L, 17% in A158T). Despite this large effect, transcriptional activation in cultured cells was only reduced to 26% in F154L and 62% in A158T of wild type activity, suggesting that a small loss of SOX9 transactivation activity could be sufficient to disrupt proper regulation of target genes during bone and testis formation. Thus, clinically relevant mutations of SOX9 affect protein structure leading to compound effects of reduced nuclear import and reduced DNA binding, the net effect being loss of transcriptional activation.

Abnormalities, Multiple↗
PubMed2001

Molecular dissection of the importin beta1-recognized nuclear targeting signal of parathyroid hormone-related protein.

Produced by various types of solid tumors, parathyroid hormone-related protein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin beta1-mediated pathway. The present study uses several different direct binding assays to map the interaction of PTHrP with importin beta using a series of alanine mutated PTHrP peptides and truncated human importin beta1 derivatives. Our results indicate that PTHrP amino acids 83-93 (KTPGKKKKGK) are absolutely essential for importin beta1 recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of importin beta1 are required for the interaction. Binding of importin beta1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein Ran, consistent with the idea that RanGTP binding to importin beta is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich importin beta1-recognized nuclear targeting signal.

Active Transport, Cell Nucleus↗
PubMed2001

Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16.

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

Active Transport, Cell Nucleus↗
PubMed2001

Roles of cytoskeletal and junctional plaque proteins in nuclear signaling.

Cytoplasmic junctional plaque proteins play an important role at intercellular junctions. They link transmembrane cell adhesion molecules to components of the cytoskeleton, thereby playing an important role in the control of many cellular processes. Recent studies on the subcellular distribution of some plaque proteins have revealed that a number of these proteins are able to localize in the nucleus. This dual location indicates that in addition to promoting adhesive interactions, plaque proteins may also play a direct role in nuclear processes, and in particular in the transfer of signals from the membrane to the nucleus. Therefore, translocation of plaque proteins into the nucleus in response to extracellular signals could represent a novel and direct mechanism by which signals can be transmitted from the plasma membrane to the nucleus. This could allow cells to respond to changing environmental conditions in a rapid and efficient way. In addition, conditional sequestration of karyophilic proteins at the sites of cell-cell and cell-substratum adhesion may represent a general mechanism for the regulation of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗
PubMed2001

Dynamics of leptomycin B-sensitive nucleocytoplasmic flux of parathyroid hormone-related protein.

Parathyroid hormone-related protein is responsible for hypercalcemia induced by various tumors. The similarity of its N-terminus to that of parathyroid hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. We have previously shown that parathyroid hormone-related protein nuclear import is mediated by importin beta1. Here we use fluorescence recovery after photobleaching for the first time to show that, in living cells, parathyroid hormone-related protein is exported from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomycin B treatment significantly reduced the rate of nuclear export 4 -10-fold, thereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold reduced nuclear import rate. Inhibiting the nuclear export of a protein able to shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization of a protein containing distinct nuclear import and export signals is the product of an integrated system. Although there have been several recent studies examining the dynamics of intranuclear transport using fluorescence recovery after photobleaching, this represents, to our knowledge, the first use of the technique to examine the kinetics of nucleocytoplasmic flux in living cells.

Animals↗
PubMed2001

Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway.

Efficient nuclear delivery of plasmid DNA represents a major barrier in nonviral gene transfer. One approach has been to use DNA-binding proteins such as GAL4 from yeast as DNA carriers with nuclear targeting properties. We recently showed, however, that GAL4 is inefficient in targeting DNA to the nucleus because its DNA-binding and nuclear targeting activities are mutually exclusive, which relates to the fact that GAL4 nuclear import occurs via a novel pathway. Here, we 'switch' this pathway to a more conventional one by adding a modified poly-lysine to which an optimized nuclear targeting signal, based on that of the SV40 large T-antigen, is linked. We also use a chimeric GAL4-alpha-melanocyte stimulating hormone (MSH) fusion protein to enable gene transfer to cells expressing the MSH receptor. Switching the nuclear import pathway of the transfecting complex significantly enhances receptor-mediated gene transfer through enabling interaction with desired components of the cellular nuclear import machinery. The present study represents the first demonstration that nuclear targeting signals can enhance receptor-mediated gene delivery, the approaches having important relevance to research and clinical applications, such as in generating transgenic or knock-out animals, or in gene therapy.

Active Transport, Cell Nucleus↗
PubMed2001

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Amino Acid Sequence↗