Classical Nuclear Import Pathway Research Articles

Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344-28354 (2020); and M. Frieman et al., J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

Molecular Mechanisms of Nuclear Transport of the Neuronal Voltage-gated Ca2+ Channel β3 Auxiliary Subunit

Previous studies have shown that in addition to its role within the voltage-gated calcium channel complex in the plasma membrane, the neuronal CaVβ subunit can translocate to the cell nucleus. However, little is known regarding the role this protein could play in the nucleus, nor the molecular mechanism used by CaVβ to enter this cell compartment. This report shows evidence that CaVβ3 has nuclear localization signals (NLS) that are not functional, suggesting that the protein does not use a classical nuclear import pathway. Instead, its entry into the nucleus could be associated with another protein that would function as a carrier, using a mechanism known as a piggyback. Mass spectrometry assays and bioinformatic analysis allowed the identification of proteins that could be participating in the entry of CaVβ3 into the nucleus. Likewise, through proximity ligation assays (PLA), it was found that members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) and B56δ, a regulatory subunit of the protein phosphatase 2A (PP2A), could function as proteins that regulate this piggyback mechanism. On the other hand, bioinformatics and site-directed mutagenesis assays allowed the identification of a functional nuclear export signal (NES) that controls the exit of CaVβ3 from the nucleus, which would allow the completion of the nuclear transport cycle of the protein. These results reveal a novel mechanism for the nuclear transport cycle of the neuronal CaVβ3 subunit.

Suppression of classical nuclear import pathway by importazole and ivermectin inhibits rotavirus replication.

Rotavirus is the foremost cause of acute gastroenteritis among infants in resource-poor countries, causing severe morbidity and mortality. The currently available rotavirus vaccines are effective in reducing severity of the disease but not the infection rates, thus antivirals as an adjunct therapy are needed to reduce the morbidity in children. Viruses rely on host cellular machinery for nearly every step of the replication cycle. Therefore, targeting host factors that are indispensable for virus replication could be a promising strategy. To assess the therapeutic potential of ivermectin and importazole against rotaviruses. Antirotaviral activity of importazole and ivermectin was measured against various rotavirus strains (RV-SA11, RV-Wa, RV-A5-13, RV-EW) in vitro and in vivo by quantifying viral protein expression by western blot, analysing viroplasm formation by confocal microscopy, and measuring virus yield by plaque assay. Importin-β1 and Ran were found to be induced during rotavirus infection. Knocking down importin-β1 severely impaired rotavirus replication, suggesting a critical role for importin-β1 in the rotavirus life cycle. In vitro studies revealed that treatment of ivermectin and importazole resulted in reduced synthesis of viral proteins, diminished production of infectious virus particles, and decrease in viroplasm-positive cells. Mechanistic study proved that both drugs perform antirotavirus activity by inhibiting the function of importin-β1. In vivo investigations in mice also confirmed the antirotavirus potential of importazole and ivermectin at non-toxic doses. Treatments of rotavirus-infected mice with either drug resulted in diminished shedding of viral particles in the stool sample, reduced expression of viral protein in the small intestine and restoration of damaged intestinal villi comapared to untreated infected mice. The study highlights the potential of importazole and ivermectin as antirotavirus therapeutics.

Structural characterization of human importin alpha 7 in its cargo-free form at 2.5\xa0\xc5 resolution

Shuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure–function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

Structural and calorimetric studies reveal specific determinants for the binding of a high-affinity NLS to mammalian importin-alpha.

The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its nuclear localization sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high-affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3, the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.

Host Cell Factors That Interact with Influenza Virus Ribonucleoproteins.

Influenza viruses hijack host cell factors at each stage of the viral life cycle. After host cell entry and endosomal escape, the influenza viral ribonucleoproteins (vRNPs) are released into the cytoplasm where the classical cellular nuclear import pathway is usurped for nuclear translocation of the vRNPs. Transcription takes place inside the nucleus at active host transcription sites, and cellular mRNA export pathways are subverted for export of viral mRNAs. Newly synthesized RNP components cycle back into the nucleus using various cellular nuclear import pathways and host-encoded chaperones. Replication of the negative-sense viral RNA (vRNA) into complementary RNA (cRNA) and back into vRNA requires complex interplay between viral and host factors. Progeny vRNPs assemble at the host chromatin and subsequently exit from the nucleus-processes orchestrated by sets of host and viral proteins. Finally, several host pathways appear to play a role in vRNP trafficking from the nuclear envelope to the plasma membrane for egress.

Inhibition of Human Adenovirus Replication by the Importin α/β1 Nuclear Import Inhibitor Ivermectin.

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

Piggybacking on Classical Import and Other Non-Classical Mechanisms of Nuclear Import Appear Highly Prevalent within the Human Proteome.

One of the most conserved cellular pathways among eukaryotes is the extensively studied classical protein nuclear import pathway mediated by importin-α. Classical nuclear localization signals (cNLSs) are recognized by importin-α and are highly predictable due to their abundance of basic amino acids. However, various studies in model organisms have repeatedly demonstrated that only a fraction of nuclear proteins contain identifiable cNLSs, including those that directly interact with importin-α. Using data from the Human Protein Atlas and the Human Reference Interactome, and proteomic data from BioID/protein-proximity labeling studies using multiple human importin-α proteins, we determine that nearly 50% of the human nuclear proteome does not have a predictable cNLS. Surprisingly, between 25% and 50% of previously identified human importin-α cargoes do not have predictable cNLS. Analysis of importin-α cargo without a cNLS identified an alternative basic rich motif that does not resemble a cNLS. Furthermore, several previously suspected piggybacking proteins were identified, such as those belonging to the RNA polymerase II and transcription factor II D complexes. Additionally, many components of the mediator complex interact with at least one importin-α, yet do not have a predictable cNLS, suggesting that many of the subunits may enter the nucleus through an importin-α-dependent piggybacking mechanism.

Interactome of the Autoimmune Risk Protein ANKRD55.

The ankyrin repeat domain-55 (ANKRD55) gene contains intronic single nucleotide polymorphisms (SNPs) associated with risk to contract multiple sclerosis, rheumatoid arthritis or other autoimmune disorders. Risk alleles of these SNPs are associated with higher levels of ANKRD55 in CD4+ T cells. The biological function of ANKRD55 is unknown, but given that ankyrin repeat domains constitute one of the most common protein-protein interaction platforms in nature, it is likely to function in complex with other proteins. Thus, identification of its protein interactomes may provide clues. We identified ANKRD55 interactomes via recombinant overexpression in HEK293 or HeLa cells and mass spectrometry. One hundred forty-eight specifically interacting proteins were found in total protein extracts and 22 in extracts of sucrose gradient-purified nuclei. Bioinformatic analysis suggested that the ANKRD55-protein partners from total protein extracts were related to nucleotide and ATP binding, enriched in nuclear transport terms and associated with cell cycle and RNA, lipid and amino acid metabolism. The enrichment analysis of the ANKRD55-protein partners from nuclear extracts is related to sumoylation, RNA binding, processes associated with cell cycle, RNA transport, nucleotide and ATP binding. The interaction between overexpressed ANKRD55 isoform 001 and endogenous RPS3, the cohesins SMC1A and SMC3, CLTC, PRKDC, VIM, β-tubulin isoforms, and 14-3-3 isoforms were validated by western blot, reverse immunoprecipitaton and/or confocal microscopy. We also identified three phosphorylation sites in ANKRD55, with S436 exhibiting the highest score as likely 14-3-3 binding phosphosite. Our study suggests that ANKRD55 may exert function(s) in the formation or architecture of multiple protein complexes, and is regulated by (de)phosphorylation reactions. Based on interactome and subcellular localization analysis, ANKRD55 is likely transported into the nucleus by the classical nuclear import pathway and is involved in mitosis, probably via effects associated with mitotic spindle dynamics.

Mechanisms of unprimed and dexamethasone-primed nonviral gene delivery to human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti‐inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX‐mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX‐mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX‐mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX‐priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.

Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation.

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in O. tsutsugamushi infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. O. tsutsugamushi antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen’s ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin β1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin β1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin β1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which O. tsutsugamushi modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin β1 and exportin 1 to antagonize a critical arm of the antimicrobial response.

Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin β, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.

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.

Nuclear transport of fungal transcription factors mediated by Importin‐α and specificities of this interaction

Importin‐α (Impα) is a protein that mediates nuclear import of macromolecules via recognition of nuclear localization sequences in the macromolecule that will be transported. Some proteins and transcription factors related with nitrogen and glycogen metabolism in fungus possess putative NLSs, suggesting that they can be transported into the nucleus via the classical nuclear import pathway, which is mediated by Impα and Importin‐β. This work presents biophysical and functional experiments that confirms the interaction between Impα from Neurospora crassa (NcImpα) and Mus musculus (MmImpα) complexed with potential NLSs of transcription factors NIT2; NCU1629 and PACC. Calorimetric analyses showed a high affinity of the NIT2_NLS, PACC_NLS and 1629_NLS peptides at the binding sites of NcImpα and a lower affinity with MmImpα. NcImpα/NIT2_NLS and MmImpα/NIT2_NLS complexes were cocrystallized and X‐ray diffraction data were collected with resolution of 2.5 and 1.5 Å, respectively. The final NcImpα/NIT2_NLS structure allowed unambiguously to observe that the NIT2_NLS peptide binds at major and minor binding sites of NcImpα. However, the final MmImpα/NIT2_NLS structure, showed the binding of the peptide just at the minor binding site. Nuclear import assays were performed with the potential NLS peptides conjugated with bovine serum albumin. Dextran was used to confirm the permeabilization of the cytoplasmatic membrane of cells by digitonin, without damaging the nuclear envelope. The nuclear import assays with NIT2‐NLS‐BSA‐Cy3 and 1629‐NLS‐BSA‐Cy3 indicated that a significant amount of these complexes were imported into the nucleus in digitonin‐permeabilized cells, incubated with cytosol from rabbit and an energy regenerating system. However the nuclear import was more efficient when a mammalian peptide was used, as confirmed by the quantification performed with Image J. The experiments confirmed that the peptides are functional NLSs, with high affinity to Impα and evidenced specificities of the interaction between NLS peptides with Impα from different organisms.Support or Funding InformationFAPESP ‐ São Paulo State Foundation

The effect of nanoparticle size and NLS density on nuclear targeting in cancer and normal cells; impaired nuclear import and aberrant nanoparticle intracellular trafficking in glioma

The cell nucleus is an interesting target in many diseases with particular interest in cancer. Previously, nuclear targeted small and large chitosan nanoparticles (S-NPs≈25nm, and L-NPs≈150nm respectively), modified with low, intermediate and high densities of NLS (L-NLS, I-NLS and H-NLS) were developed and assessed in L929 fibroblasts. However, to evade apoptosis and stimulate tumor growth cancer cells are capable of manipulating the nuclear-cytoplasmic transport on many levels, making NPs that are capable of nuclear targeting in normal cells incapable of doing so in cancer. For such reason, here, the nuclear delivery efficiency of S-NPs and L-NPs was assessed as a function of their NLS density in cancer and non-cancer cells. For S-NPs, in all cells tested, NLS was unnecessary for nuclear delivery; unmodified S-NPs showed higher nuclear delivery than NLS-S-NPs due to their ability to gain nuclear entry in a passive manner. For L-NPs, L-NLS-L-NPs showed ≈ 8.5, 33, 1.8 and 7.2 fold higher nuclear deliveries than H-NLS-L-NPs in L929 fibroblasts, primary human fibroblasts, HEK 293 and lung cancer cells, respectively. In glioma however, unmodified L-NPs showed highest nuclear delivery, whereas NLS-L-NPs were retained in the cytoplasm. Experiments conducted in the presence of inhibitors of the classical nuclear import pathway indicated that due to overexpression of importin α, classical nuclear import in glioma is impaired leading to aberrant NP intracellular trafficking and nuclear import.

Bending-Twisting Motions and Main Interactions in Nucleoplasmin Nuclear Import.

Alpha solenoid proteins play a key role in regulating the classical nuclear import pathway, recognizing a target protein and transporting it into the nucleus. Importin-α (Impα) is the solenoid responsible for cargo protein recognition, and it has been extensively studied by X-ray crystallography to understand the binding specificity. To comprehend the main motions of Impα and to extend the information about the critical interactions during carrier-cargo recognition, we surveyed different conformational states based on molecular dynamics (MD) and normal mode (NM) analyses. Our model of study was a crystallographic structure of Impα complexed with the classical nuclear localization sequence (cNLS) from nucleoplasmin (Npl), which was submitted to multiple 100 ns of MD simulations. Representative conformations were selected for calculating the 87 lowest frequencies NMs of vibration, and a displacement approach was applied along each NM. Based on geometric criteria, using the radius of curvature and inter-repeat angles as the reference metrics, the main motions of Impα were described. Moreover, we determined the salt bridges, hydrogen bonds and hydrophobic interactions in the Impα-NplNLS interface. Our results show the bending and twisting motions participating in the recognition of nuclear proteins, allowing the accommodation and adjustment of a classical bipartite NLS sequence. The essential contacts for the nuclear import were also described and were mostly in agreement with previous studies, suggesting that the residues in the cNLS linker region establish important contacts with Impα adjusting the cNLS backbone. The MD simulations combined with NM analysis can be applied to the Impα-NLS system to help understand interactions between Impα and cNLSs and the analysis of non-classic NLSs.

Structural and Calorimetric Studies Demonstrate that Xeroderma Pigmentosum Type G (XPG) Can Be Imported to the Nucleus by a Classical Nuclear Import Pathway via a Monopartite NLS Sequence

Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin‐α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG.

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

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe 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 viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal.

Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.

Importin alpha and nonclassical nuclear localization signal

In the classical nuclear import pathway, the specific recognition between the nuclear receptor (importin-α) and the nuclear localization signals (NLSs) plays an essential role on facilitating the cargo import process. Importin-α has two separate NLS-binding sites (the major and the minor sites), accommodate NLSs, comprising of one (monopartite) or two clusters (bipartite) of basic residues connected by a 10 - 12 residue linker. The major NLS-binding site is the preferential binding site for most of the monopartite NLSs characterized to date. By screening random peptide libraries using importin-α variants as bait, the bound NLS sequences could be divided into six classes [1]. The class-3 minor site-specific NLSs and class-5 plant-specific NLSs feature a shorter basic cluster. The molecular basis of the specific binding between these non-classical NLSs and importin-α was not known and in particular, there was a lack of crystal structures of plant importin-α. Here, we present the first crystal structure of plant importin-α, and explain the differential binding specificity between the class-5 plant-specific NLSs and importin-α variants [2]. The binding conformation of the class-3 minor site-specific NLSs features an α-helical turn, that is distinct from the other NLSs reported structurally [3]. Comparative bioinformatic screens not only indicate both plant-specific and minor site-specific NLSs are much less prevalent than the classical NLSs, but also reveal a greater prevalence of these two classes of non-classical NLSs in rice the proteome, compared to the others from yeast, mammals, and even other plant species. Together, our data can help to characterize novel proteins containing non-classical NLSs destined for the cell nucleus by the classical nuclear import pathway.