Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/ß Nuclear Import Pathway.

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/ß-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/ß-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/ß nuclear import pathway.

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing.

Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.

Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1ß (HNF1ß) transcription factor is imported into the nucleus via a monopartite NLS sequence.

The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1ß transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1ß and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1ß interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1ß (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1ß NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1ß and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1ß via its nuclear import pathway.