The Experts below are selected from a list of 1146 Experts worldwide ranked by ideXlab platform
David A. Jans - One of the best experts on this subject based on the ideXlab platform.
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Sequence variation and conformational flexibility of NS5 Cter18 regulates the oligomeric state, nuclear localization, viral replication.
2016Co-Authors: Moon Y. F. Tay, David A. Jans, Kate Smith, Kitti W. K. Chan, Yongqian Zhao, Eng Eong Ooi, Julien Lescar, Dahai Luo, Jade K ForwoodAbstract:(A) Sequence variation of residues 828–848 and 883–900 of DENV1–4 and representative flaviviruses. Arrow highlights the completely conserved Y838 and R888. The alignment was performed using Clustal Omega. The virus sequences and their GenBank accession numbers are as follows: DENV1-4 (same GenBank accession number as above), Japanese Encephalitis virus (JEV; M55506), West Nile virus (WNV; M12294), Murray Valley Encephalitis virus (MVE; AF161266), Tick-Borne Encephalitis Virus (TBEV; U27495), Yellow Fever virus (YFV; X15062) and Zika virus (ZIKV;KU497555) [58]. (B) Model of the role of the C-terminal region of NS5 that is required for the formation of dimer and the initiation of de novo viral RNA replication and interaction with importin-α or NS3. On the left: de novo RNA synthesis by NS5 oligomers independent of NS3 interaction as implied by NS3 N570A mutation [59] and non-viability of NS5 R888A as identified in this study. On the right: NS3 interaction with NS5 thumb domain in the RC will support higher plus strand RNA synthesis or NS5 Cter18 interacts with host Importins or other factors to be localized to nucleus or cytoplasm in a regulated manner.
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nuclear localization of the dystrophin associated protein α dystrobrevin through importin α2 β1 is critical for interaction with the nuclear lamina maintenance of nuclear integrity
The FASEB Journal, 2015Co-Authors: Areli Aguilar, Kylie M. Wagstaff, Samuel Zinker, Rocio Suarezsanchez, David A. JansAbstract:Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific Importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased acc...
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Putting things in place for fertilization: discovering roles for importin proteins in cell fate and spermatogenesis
Wolters Kluwer Medknow Publications, 2015Co-Authors: Kate L Loveland, David A. Jans, Andrew T. Major, Romaly Butler, Julia C. Young, Yoichi MiyamotoAbstract:Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the Importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses
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nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce venezuelan equine encephalitis virus replication
Antiviral Research, 2013Co-Authors: Lindsay Lundberg, David A. Jans, Kylie M. Wagstaff, Chelsea Pinkham, Alan Baer, Moushimi Amaya, Aarthi Narayanan, Kylene KehnhallAbstract:Targeting host responses to invading viruses has been the focus of recent antiviral research. Venezuelan Equine Encephalitis Virus (VEEV) is able to modulate host transcription and block nuclear trafficking at least partially due to its capsid protein forming a complex with the host proteins importin α/β1 and CRM1. We hypothesized that disrupting the interaction of capsid with importin α/β1 or the interaction of capsid with CRM1 would alter capsid localization, thereby lowering viral titers in vitro. siRNA mediated knockdown of importin α, importin β1, and CRM1 altered capsid localization, confirming their role in modulating capsid trafficking. Mifepristone and ivermectin, inhibitors of importin α/β-mediated import, were able to reduce nuclear-associated capsid, while leptomycin B, a potent CRM1 inhibitor, confined capsid to the nucleus. In addition to altering the level and distribution of capsid, the three inhibitors were able to reduce viral titers in a relevant mammalian cell line with varying degrees of efficacy. The inhibitors were also able to reduce the cytopathic effects associated with VEEV infection, hinting that nuclear import inhibitors may be protecting cells from apoptosis in addition to disrupting the function of an essential viral protein. Our results confirm that VEEV uses host Importins and exportins during part of its life cycle. Further, it suggests that temporarily targeting host proteins that are hijacked for use by viruses is a viable antiviral therapy.
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nuclear transport of parathyroid hormone pth related protein is dependent on microtubules
Molecular Endocrinology, 2002Co-Authors: David A. Jans, Kate L Loveland, Mark H C Lam, Rachel J Thomas, Steven Schilders, John T Martin, Matthew T GillespieAbstract:PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin β1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin β1, but not importinα , enhanced the microtubular associa...
Stephane Boivin - One of the best experts on this subject based on the ideXlab platform.
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large scale conformational dynamics control h5n1 influenza polymerase pb2 binding to importin α
Journal of the American Chemical Society, 2015Co-Authors: Stephane Boivin, Elise Delaforge, Sigrid Milles, Guillaume Bouvignies, Denis Bouvier, Nicola SalviAbstract:Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for Importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Forster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin alpha and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin alpha. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.
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interaction of the influenza a virus polymerase pb2 c terminal region with importin α isoforms provides insights into host adaptation and polymerase assembly
Journal of Biological Chemistry, 2011Co-Authors: Stephane Boivin, Darren J HartAbstract:In the adaptation of avian viruses to mammalian hosts, mutations in the viral polymerase, notably in the PB2 subunit, play an important role. A PB2 C-terminal domain rich in putative host adaptation residues has been shown to bind importin α nuclear import receptors. Adaptation has been proposed to involve binding of PB2 to Importins of the new host. To date PB2-importin complexes have been characterized semiquantitatively with no precise measurement of binding parameters. To investigate the effects of adaptive mutations on importin interaction and selectivity, surface plasmon resonance was used to compare the binding rate constants and affinities of avian H5N1 and human H3N2 PB2 C-terminal variants with importin isoforms human α 1, 3, 5 and 7, and avian α 1. Using purified proteins eliminates host environment effects and permits measurement of intrinsic affinities and rates of complex formation and dissociation. Two effects were observed: first, adaptive mutations D701N, R702K, and S714R in the nuclear localization signal domain increased 2–4-fold the association rates with avian and human Importins; second, measurement of different structural forms of the PB2 C terminus demonstrated that the upstream 627 domain reduced binding affinity, consistent with a steric clash predicted from crystal structures. From these kinetic data, structural analyses, and the data of others, a model is proposed in which an increase in charged surface residues during host adaptation increases the association rate of PB2 to cytoplasmic Importins and where the C-terminal 627-nuclear localization signal domain may reorganize upon importin binding, consistent with a role in active polymerase assembly.
Elise Delaforge - One of the best experts on this subject based on the ideXlab platform.
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large scale conformational dynamics control h5n1 influenza polymerase pb2 binding to importin α
Journal of the American Chemical Society, 2015Co-Authors: Stephane Boivin, Elise Delaforge, Sigrid Milles, Guillaume Bouvignies, Denis Bouvier, Nicola SalviAbstract:Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for Importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Forster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin alpha and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin alpha. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.
Nicola Salvi - One of the best experts on this subject based on the ideXlab platform.
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large scale conformational dynamics control h5n1 influenza polymerase pb2 binding to importin α
Journal of the American Chemical Society, 2015Co-Authors: Stephane Boivin, Elise Delaforge, Sigrid Milles, Guillaume Bouvignies, Denis Bouvier, Nicola SalviAbstract:Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for Importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Forster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin alpha and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin alpha. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.
Sigrid Milles - One of the best experts on this subject based on the ideXlab platform.
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large scale conformational dynamics control h5n1 influenza polymerase pb2 binding to importin α
Journal of the American Chemical Society, 2015Co-Authors: Stephane Boivin, Elise Delaforge, Sigrid Milles, Guillaume Bouvignies, Denis Bouvier, Nicola SalviAbstract:Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for Importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Forster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin alpha and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin alpha. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.
