43 research outputs found

    Diversification of importin-α isoforms in cellular trafficking and disease states.

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    The human genome encodes seven isoforms of importin α which are grouped into three subfamilies known as α1, α2 and α3. All isoforms share a fundamentally conserved architecture that consists of an N-terminal, autoinhibitory, importin-β-binding (IBB) domain and a C-terminal Arm (Armadillo)-core that associates with nuclear localization signal (NLS) cargoes. Despite striking similarity in amino acid sequence and 3D structure, importin-α isoforms display remarkable substrate specificity in vivo. In the present review, we look at key differences among importin-α isoforms and provide a comprehensive inventory of known viral and cellular cargoes that have been shown to associate preferentially with specific isoforms. We illustrate how the diversification of the adaptor importin α into seven isoforms expands the dynamic range and regulatory control of nucleocytoplasmic transport, offering unexpected opportunities for pharmacological intervention. The emerging view of importin α is that of a key signalling molecule, with isoforms that confer preferential nuclear entry and spatiotemporal specificity on viral and cellular cargoes directly linked to human diseases

    Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

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    Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs through the nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study, it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B virus core protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLS by co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion protein were expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. The results indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolated from E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transport study shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc and rHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm into the nucleus

    Molecular characterization of Lhx8, an oocyte-specific transcription factor, and its interacting proteins in rainbow trout and cattle

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    LIM homeobox 8 (Lhx8) is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. To date, no attempts have been made to investigate the existence of any cellular factors that might interact with Lhx8 protein in oocytes and early embryos. In this study, we report the characterization of rainbow trout and bovine Lhx8 genes and identification of important germ cell-specific nuclear factors that interact with Lhx8 protein in both species. In rainbow trout, two Lhx8 genes, Lhx8a and Lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout. The open reading frame (ORF) of bovine Lhx8 gene was amplified from cDNA of a bovine fetal ovary using primers designed based on a predicted bovine Lhx8 cDNA sequence and a partial 5 \u27end transcript. The ORF of bovine Lhx8 cDNA is 1,134 bp in length encoding a protein of 377 amino acids. A splicing variant of Lhx8 (Lhx8_v1) was identified, which results from alternative splicing of exon 2 and 3, and encodes a protein of 293 amino acids. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in the splicing variant, Lhx8_v1, is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 mRNA are specifically expressed in fetal ovaries and testis but not detectable in the somatic tissues as well as in granulosa and theca cells. Lhx8 mRNA is highly abundant in GV and MII stage oocytes as well as in early stage embryos but not detectable in morula and blastocyst stage embryos. Lhx8_v1 mRNA expression is detectable in oocytes and early embryo but not in morula and blastocyst stage embryos. A GFP reporter assay revealed that Lhx8 is a nuclear protein and the predicted monopartite NLS is required for its transport into the nucleus. Direct yeast two-hybrid analysis revealed that bovine Lhx8 protein interacts with Figla, a basic helix-loop-helix transcription factor. The interaction between Lhx8 and Figla was confirmed by a co-immunoprecipitation assay. This is the first time that a direct protein-protein interaction between two germ cell-specific transcription factors essential for oocyte and follicular development is demonstrated. The study provides new information for studying the mechanisms of the regulatory roles of Lhx8 in oocyte/follicular development and early embryogenesis

    The importin-β binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro

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    The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-β binding (IBB) domain and a COOH-terminal m3G-cap–binding region that bears no structural similarity to known import adaptors like importin-α (impα). Here, we show that recombinant snurportin1 and importin-β (impβ) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impα–dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1–impβ, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impβ and a snurportin1 mutant containing an impα IBB domain (IBBimpα), nuclear U1 snRNP import was Ran dependent. Furthermore, β-galactosidase (βGal) containing a snurportin1 IBB domain, but not IBBimpα-βGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impβ with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions

    Differential use of importin-α isoforms governs cell tropism and host adaptation of influenza virus

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    Influenza A viruses are a threat to humans due to their ability to cross species barriers, as illustrated by the 2009 H1N1v pandemic and sporadic H5N1 transmissions. Interspecies transmission requires adaptation of the viral polymerase to importin-α, a cellular protein that mediates transport into the nucleus where transcription and replication of the viral genome takes place. In this study, we analysed replication, host specificity and pathogenicity of avian and mammalian influenza viruses, in importin-α-silenced cells and importin-α-knockout mice, to understand the role of individual importin-α isoforms in adaptation. For efficient virus replication, the polymerase subunit PB2 and the nucleoprotein (NP) of avian viruses required importin-α3, whereas PB2 and NP of mammalian viruses showed importin-α7 specificity. H1N1v replication depended on both, importin-α3 and -α7, suggesting ongoing adaptation of this virus. Thus, differences in importin-α specificity are determinants of host range underlining the importance of the nuclear envelope in interspecies transmission
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