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Yoshihiro Yoneda - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of importin α8 as a classical Nuclear Localization Signal receptor
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015Co-Authors: Chihiro Kimoto, Masahiro Oka, Yoichi Miyamoto, Tetsuji Moriyama, Akira Tsujii, Yoshinobu Igarashi, Chikashi Obuse, Yoshihiro YonedaAbstract:Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin β1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the Nuclear transport of classical Nuclear Localization Signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin β1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/β1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin β binding domain.
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zinc finger domain of snail functions as a Nuclear Localization Signal for importin beta mediated Nuclear import pathway
Genes to Cells, 2005Co-Authors: Hideki Yamasaki, Toshihiro Sekimoto, Tadashi Ohkubo, Tsutomu Douchi, Yukihiro Nagata, Masayuki Ozawa, Yoshihiro YonedaAbstract:Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid Nuclear protein with an amino-terminal basic amino acid-rich domain (SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). A series of fusion proteins composed of green fluorescent protein (GFP) and portions of the Snail protein were generated, and their subcellular Localization was examined. Fusion of the four zinc fingers to GFP led to the targeting of GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for Nuclear Localization. Using an in vitro transport system, the Nuclear import of Snail was reconstituted by importin (karyopherin) beta in the presence of Ran and NTF2. We further demonstrated that Snail binds directly to importin beta in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a Nuclear Localization Signal and Snail can be transported into the nucleus in an importin beta-mediated manner.
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differential modes of Nuclear Localization Signal nls recognition by three distinct classes of nls receptors
Journal of Biological Chemistry, 1997Co-Authors: Yoichi Miyamoto, Naoko Imamoto, Toshihiro Sekimoto, Taro Tachibana, Takehiko Seki, Shusuke Tada, Takemi Enomoto, Yoshihiro YonedaAbstract:The targeting of karyophilic proteins to Nuclear pores is mediated via the formation of a Nuclear pore-targeting complex, through the interaction of Nuclear Localization Signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the Nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.
Gino Cingolani - One of the best experts on this subject based on the ideXlab platform.
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divergent evolution of Nuclear Localization Signal sequences in herpesvirus terminase subunits
Journal of Biological Chemistry, 2016Co-Authors: Rajeshwer S Sankhala, Ravi K Lokareddy, Gino CingolaniAbstract:The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host Nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of Nuclear Localization Signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the β-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. We propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase Nuclear import, reflecting a mechanism of virus:host adaptation.
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phospholipid scramblase 1 contains a nonclassical Nuclear Localization Signal with unique binding site in importin α
Journal of Biological Chemistry, 2005Co-Authors: Minhsuan Chen, Iris Benefraim, Gregory Mitrousis, Nancy Walkerkopp, Peter J Sims, Gino CingolaniAbstract:Nuclear import of proteins containing a classical Nuclear Localization Signal (NLS) is an energy-dependent process that requires the heterodimer importin α/β. Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257GKISKHWTGI266. This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 A crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin α. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1–4 of importin α, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel Nuclear Localization Signal that presents a distinct motif for binding to importin α.
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Molecular Basis for the Recognition of a Nonclassical Nuclear Localization Signal by Importin β
Molecular cell, 2002Co-Authors: Gino Cingolani, Janna Bednenko, Matthew T. Gillespie, Larry GeraceAbstract:Abstract Nuclear import of proteins containing a classical Nuclear Localization Signal (NLS) involves NLS recognition by importin α, which associates with importin β via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin β. We solved the crystal structure of a fragment of importin β-1 (1–485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin β distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin β (1–485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical Nuclear import receptor domain, which could have evolved into the modern importin β superfamily.
Sanjeeb K. Sahoo - One of the best experts on this subject based on the ideXlab platform.
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intracellular trafficking of Nuclear Localization Signal conjugated nanoparticles for cancer therapy
European Journal of Pharmaceutical Sciences, 2010Co-Authors: Ranjita Misra, Sanjeeb K. SahooAbstract:Abstract Doxorubicin (DOX) is an anticancer drug with an intracellular site of action in the nucleus. For high antitumour activity, it should be effectively internalized into the cancer cells and accumulate in the nucleus. In this study, we have prepared a Nuclear Localization Signal conjugated doxorubicin loaded Poly ( d , l -lactide-co-glycolide) nanoparticles (NPs), to deliver doxorubicin to the nucleus efficiently. Physico-chemical characterization of these NPs showed that the drug is molecularly dispersed in spherical and smooth surfaced nanoparticles. NPs (∼226 nm in diameter, 46% encapsulation efficiency) under in vitro conditions exhibited sustained release of the encapsulated drug (63% release in 60 days). Cell cytotoxicity results showed that NLS conjugated NPs exhibited comparatively lower IC50 value (2.3 μM/ml) than drug in solution (17.6 μM/ml) and unconjugated NPs (7.9 μM/ml) in breast cancer cell line MCF-7 as studied by MTT assay. Cellular uptake studies by confocal laser scanning microscopy (CLSM) and fluorescence spectrophotometer showed that greater amount of drug is targeted to the nucleus with NLS conjugated NPs as compared to drug in solution or unconjugated NPs. Flow cytometry experiments results showed that NLS conjugated NPs are showing greater cell cycle (G2/M phase) blocking and apoptosis than native DOX and unconjugated NPs. In conclusion, these results suggested that NLS conjugated doxorubicin loaded NPs could be potentially useful as novel drug delivery system for breast cancer therapy.
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Intracellular trafficking of Nuclear Localization Signal conjugated nanoparticles for cancer therapy.
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2009Co-Authors: Ranjita Misra, Sanjeeb K. SahooAbstract:Doxorubicin (DOX) is an anticancer drug with an intracellular site of action in the nucleus. For high antitumour activity, it should be effectively internalized into the cancer cells and accumulate in the nucleus. In this study, we have prepared a Nuclear Localization Signal conjugated doxorubicin loaded Poly (D,L-lactide-co-glycolide) nanoparticles (NPs), to deliver doxorubicin to the nucleus efficiently. Physico-chemical characterization of these NPs showed that the drug is molecularly dispersed in spherical and smooth surfaced nanoparticles. NPs (approximately 226 nm in diameter, 46% encapsulation efficiency) under in vitro conditions exhibited sustained release of the encapsulated drug (63% release in 60 days). Cell cytotoxicity results showed that NLS conjugated NPs exhibited comparatively lower IC(50) value (2.3 microM/ml) than drug in solution (17.6 microM/ml) and unconjugated NPs (7.9 microM/ml) in breast cancer cell line MCF-7 as studied by MTT assay. Cellular uptake studies by confocal laser scanning microscopy (CLSM) and fluorescence spectrophotometer showed that greater amount of drug is targeted to the nucleus with NLS conjugated NPs as compared to drug in solution or unconjugated NPs. Flow cytometry experiments results showed that NLS conjugated NPs are showing greater cell cycle (G2/M phase) blocking and apoptosis than native DOX and unconjugated NPs. In conclusion, these results suggested that NLS conjugated doxorubicin loaded NPs could be potentially useful as novel drug delivery system for breast cancer therapy.
Witold Filipowicz - One of the best experts on this subject based on the ideXlab platform.
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the double stranded rna binding domain of human dicer functions as a Nuclear Localization Signal
RNA, 2013Co-Authors: Michael Doyle, Lukas Badertscher, Lukasz Jaskiewicz, Stephan Guttinger, Sabine Jurado, Tabea Hugenschmidt, Ulrike Kutay, Witold FilipowiczAbstract:Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and double-stranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a Nuclear Localization Signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate Nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to Nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state Localization is cytoplasmic.
David S Goldfarb - One of the best experts on this subject based on the ideXlab platform.
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a Nuclear export Signal prevents saccharomyces cerevisiae hsp70 ssb1p from stimulating Nuclear Localization Signal directed Nuclear transport
Journal of Biological Chemistry, 1999Co-Authors: Nataliya Shulga, Philip James, Elizabeth A Craig, David S GoldfarbAbstract:Abstract Hsp70 has been implicated in Nuclear Localization Signal (NLS)-directed Nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSBgene families of cytosolic Hsp70s. The nucleocytoplasmic Localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich Nuclear export Signal (NES) that is necessary and sufficient to direct Nuclear export. The accumulation of GFP-Ssb1p in the nuclei ofxpo1-1 cells suggests that Ssb1p shuttles across the Nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP Nuclear Localization defects of nup188-Δ cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in Nuclear transport. Thus, NES-less Ssb1p stimulates Nuclear transport innup188-Δ cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in Nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.
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a Nuclear Localization Signal within hiv 1 matrix protein that governs infection of non dividing cells
Nature, 1993Co-Authors: Michael Bukrinsky, David S Goldfarb, Sheryl Haggerty, Michael P Dempsey, N Sharova, Alexei A Adzhubei, L Spitz, Paul Lewis, Michael Emerman, Mario StevensonAbstract:PERMISSIVENESS of the host cell to productive infection by onco-retroviruses is cell-cycle dependent1, and Nuclear Localization of viral nucleoprotein preintegration complexes will occur only after cells have passed through mitosis2. In contrast, establishment of an integrated provirus after infection by the lentivirus HIV-1 is independent of host cell proliferation3–5. The ability of HIV-1 to replicate in non-dividing cells is partly accounted for by the kary-ophilic properties of the viral preintegration complex which, after virus infection, is actively transported to the host cell nucleus. Here we report that the gag matrix protein of HIV-1 contains a Nuclear Localization sequence which, when conjugated to a heterologous protein, directs its Nuclear import. In addition, HIV-1 mutants containing amino-acid substitutions in this Nuclear Localization Signal integrate and replicate within dividing but not growth-arrested cells, and thus display a phenotype more representative of an onco-retrovirus.
