Nuclear import of LIKE HETEROCHROMATIN PROTEIN1 is redundantly mediated by importins α-1, α-2 and α-3.
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ABSTRACT:
SUBMITTER: Chen C
PROVIDER: S-EPMC7810169 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element-binding proteins through interaction with importin ?. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins ?. However, selectivity and specificity of NTMs for importins/karyopherins ? were undetermined. METHODS AND RESULTS:We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NF?B1/p50, with endogenous human importins/karyopherins ? to determine the mechanism of NTM modulation of importin ?-mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins ?. However, only interaction with importin ?5 (Imp ?5) displayed specific, high-affinity binding. The 2:1 stoichiometry of the N50-Imp ?5 interaction (KD1 = 73 nmol/L, KD2 = 140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3-dimensional (3-D) docking models and comparative structural analysis, we identified a structural component of the Imp ?5 major NLS binding pocket that may stabilize N50 binding. Imp ?5 also displayed rapid stimulus-induced turnover, which could influence its availability for nuclear transport during the inflammatory response. CONCLUSIONS:These results provide direct evidence that N50 peptide selectively targets Imp ?5, encouraging further refinement of NLS-derived peptides as new tools to modulate inflammatory disorders.
| S-EPMC3835248 | biostudies-literature
Project description:Synapse-to-nucleus signaling is critical for synaptic development and plasticity. In Drosophila, the ligand Wingless causes the C terminus of its Frizzled2 receptor (Fz2-C) to be cleaved and translocated from the postsynaptic density to nuclei. The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain. We found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large. These developmental defects in imp-beta11 mutants could be overcome by expression of Fz2-C fused to a nuclear localization sequence that can bypass Importin-beta11. Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway.
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Project description:Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.
| S-EPMC2740467 | biostudies-literature
Project description:The NAD-dependent deacetylase Sirtuin-2 (SIRT2) functions in diverse cellular processes including the cell cycle, metabolism, and has important roles in tumorigenesis and bacterial infection. SIRT2 predominantly resides in the cytoplasm but can also function in the nucleus. Consequently, SIRT2 localisation and its interacting partners may greatly impact its function and need to be defined more clearly. In this study we used mass spectrometry to determine the interactomes of SIRT2 in whole cells and in specific cellular fractions; cytoplasm, nucleus and chromatin. Using this approach, we identified novel interacting partners of SIRT2. These included a number of proteins that function in nuclear import. We show that multiple importins interact with and contribute to the basal nuclear shuttling of SIRT2 and that one of these, IPO7 is required for SIRT2 mediated H3K18 deacetylation in response to bacterial infection. Furthermore, we reveal that the unstructured C-terminus of SIRT2 negatively regulates importin-binding and nuclear transport. This study demonstrates that SIRT2 is actively transported into the nucleus via a process regulated by its C-terminus and provides a resource of SIRT2 interacting partners.
| S-EPMC7010746 | biostudies-literature
Project description:Many nuclear transport pathways are mediated by importin beta-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impbeta and their respective basic import substrates. The Impbeta-Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins.
| S-EPMC125346 | biostudies-literature
Project description:Cellular nucleocytoplasmic trafficking is mediated by the importin family of nuclear transport proteins. The well-characterized importin alpha (IMPA) and importin beta (IMPB) nuclear import pathway plays a crucial role in the innate immune response to viral infection by mediating the nuclear import of transcription factors such as IRF3, NFκB, and STAT1. The nuclear transport of these transcription factors ultimately leads to the upregulation of a wide range of antiviral genes, including IFN and IFN-stimulated genes (ISGs). To replicate efficiently in cells, viruses have developed mechanisms to block these signaling pathways. One strategy to evade host innate immune responses involves blocking the nuclear import of host antiviral transcription factors. By binding IMPA proteins, these viral proteins prevent the nuclear transport of key transcription factors and suppress the induction of antiviral gene expression. In this review, we describe examples of proteins encoded by viruses from several different families that utilize such a competitive inhibition strategy to suppress the induction of antiviral gene expression.
| S-EPMC10778312 | biostudies-literature
Project description:ARHI (aplasia Ras homologue member I; also known as DIRAS3) is an imprinted tumour suppressor gene, the expression of which is lost in the majority of breast and ovarian cancers. Unlike its homologues Ras and Rap, ARHI functions as a tumour suppressor. Our previous study showed that ARHI can interact with the transcriptional activator STAT3 (signal transducer and activator of transcription 3) and inhibit its nuclear translocation in human breast- and ovarian-cancer cells. To identify proteins that interact with ARHI in nuclear translocation, in the present study, we performed proteomic analysis and identified several importins that can associate with ARHI. To further explore this novel finding, we purified 10 GST (glutathione transferase)-importin fusion proteins (importins 7, 8, 13, beta1, alpha1, alpha3, alpha5, alpha6, alpha7 and mutant alpha1). Using a GST-pulldown assay, we found that ARHI can bind strongly to most importins; however, its binding is markedly reduced with an importin alpha1 mutant that contains an altered NLS (nuclear-localization signal) domain. In addition, an ARHI N-terminal deletion mutant exhibits greatly reduced binding to all importins compared with wild-type ARHI. In nuclear-import assays, the addition of ARHI blocked nuclear localization of phosphorylated STAT3. ARHI also inhibits the interaction of Ran-importin complexes with GFP (green fluorescent protein) fusion proteins that contain an NLS domain and a beta-like import receptor-binding domain, thereby blocking their nuclear localization. By conducting GST-pulldown assays, we found that ARHI could compete for Ran-importin binding. Thus ARHI-induced disruption of importin-binding to cargo proteins, including STAT3, could serve as an important regulatory mechanism that contributes to the tumour-suppressor function of ARHI.
| S-EPMC2974631 | biostudies-literature
Project description:The Notch signaling pathway is an important regulation system for the development and self-renewal of different tissues. A specific feature of this signaling cascade is the function of Notch as a surface receptor and regulator of gene expression. Hence, Notch activation and signal transduction requires the proteolytic release of the Notch intracellular domain (NICD), which activates the transcription of cell-specific genes after its transport into the nucleus. To date, little is known about the mechanisms that mediate NICD nuclear import. We here show that transport of NICD into the nucleus is mediated by the canonical importin alpha/beta1 pathway. GST pull-down experiments revealed that NICD binds via one of its four potential nuclear localization signals to importins alpha3, alpha4, and alpha7, but not to alpha1 and alpha5. siRNA-mediated knockdown experiments showed that importins alpha3, alpha4 (and to a lesser extent, alpha7) mediate nuclear import of NICD and thus are directly involved in Notch signaling.
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Project description:Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase-M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1-A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.
| S-EPMC2132560 | biostudies-literature
Project description:Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/β-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.
| S-EPMC8754214 | biostudies-literature