Karyopherin Beta Research Articles

A mass spectrometry-based approach for the identification of Kpnβ1 binding partners in cancer cells

Karyopherin beta 1 (Kpnβ1) is the principal nuclear importer of cargo proteins and plays a role in many cellular processes. Its expression is upregulated in cancer and essential for cancer cell viability, thus the identification of its binding partners might help in the discovery of anti-cancer therapeutic targets and cancer biomarkers. Herein, we applied immunoprecipitation coupled to mass spectrometry (IP-MS) to identify Kpnβ1 binding partners in normal and cancer cells. IP-MS identified 100 potential Kpnβ1 binding partners in non-cancer hTERT-RPE1, 179 in HeLa cervical cancer, 147 in WHCO5 oesophageal cancer and 176 in KYSE30 oesophageal cancer cells, including expected and novel interaction partners. 38 binding proteins were identified in all cell lines, with the majority involved in RNA metabolism. 18 binding proteins were unique to the cancer cells, with many involved in protein translation. Western blot analysis validated the interaction of known and novel binding partners with Kpnβ1 and revealed enriched interactions between Kpnβ1 and select proteins in cancer cells, including proteins involved in cancer development, such as Kpnα2, Ran, CRM1, CCAR1 and FUBP1. Together, this study shows that Kpnβ1 interacts with numerous proteins, and its enhanced interaction with certain proteins in cancer cells likely contributes to the cancer state.

Expression of Karyopherin Alpha 2 and Karyopherin Beta 1 Correlate with Poor Prognosis in Gastric Cancer

Introduction: Karyopherin alpha 2 (KPNA2) and karyopherin beta 1 (KPNB1) constitute nuclear transport protein complexes involved in nuclear import and are significant in tumor progression. Although high KPNA2 expression was associated with poor prognosis in solid tumors, the relationship between KPNA2 and KPNB1 expression and their prognostic role in gastric cancer (GC) remains unclear. Methods: Immunohistochemistry was used to correlate the expression of KPNA2 and KPNB1 with various features, including clinicopathological characteristics in 130 patients with GC and survival in 94 patients with invasive lesions extending to the submucosa or deeper. Results: High expression of KPNA2 and KPNB1 was found in 25% and 36% of the patients, respectively. Both were significantly related to tumor depth, lymph node metastasis, lymphatic invasion, venous invasion, and Ki-67 expression. KPNA2 expression was significantly related to that of KPNB1 (p < 0.001). Patients with high KPNB1 expression had poorer prognosis than those with low expression (p = 0.027), as was also observed in case of KPNA2 (p < 0.001). Patients with high expression of both KPNA2 and KPNB1 accounted for 18% and had a poorer prognosis than those with high expression of either and those with low expression of both (p = 0.001). Multivariate analysis revealed that high expression of both KPNA2 and KPNB1 was an independent prognostic factor in patients with GC (hazard ratio, 3.46; 95% confidence interval, 1.64–2.73, p = 0.001). Conclusion: KPNA2 expression was correlated with KPNB1 expression, and high co-expression of KPNA2 and KPNB1 may represent a strong prognostic biomarker in GC.

Novel small molecule inhibitor of Kpnβ1 induces cell cycle arrest and apoptosis in cancer cells

Karyopherin beta 1 (Kpnβ1) is a major nuclear import receptor that mediates the import of cellular cargoes into the nucleus. Recently it has been shown that Kpnβ1 is highly expressed in several cancers, and its inhibition by siRNA induces apoptotic cancer cell death, while having little effect on non-cancer cells. This study investigated the effect of a novel small molecule, Inhibitor of Nuclear Import-60 (INI-60), on cancer cell biology, as well as nuclear import activities associated with Kpnβ1, and cancer progression in vivo using cervical and oesophageal cancer cell lines. INI-60 treatment resulted in the inhibition of cancer cell proliferation, colony formation, migration and invasion, and induced a G1/S cell cycle arrest, followed by cancer cell death via apoptosis. Non-cancer cells were minimally affected by INI-60 at concentrations that inhibited cancer cells. INI-60 treatment altered the localisation of Kpnβ1 and its cargoes, NFκB/p65, NFAT and AP-1, and the overexpression of Kpnβ1 reduced INI-60 cytotoxicity. INI-60 also inhibited KYSE 30 oesophageal cancer cell line growth in vivo. Taken together, these results show that INI-60 inhibits the nuclear import of Kpnβ1 cargoes and interferes with cancer cell biology. INI-60 presents as a potential therapeutic approach for cancers of different tissue origins and warrants further investigation as a novel anti-cancer agent.

Inhibition of Kpn\u03b21 mediated nuclear import enhances cisplatin chemosensitivity in cervical cancer

BackgroundInhibition of nuclear import via Karyopherin beta 1 (Kpnβ1) shows potential as an anti-cancer approach. This study investigated the use of nuclear import inhibitor, INI-43, in combination with cisplatin.MethodsCervical cancer cells were pre-treated with INI-43 before treatment with cisplatin, and MTT cell viability and apoptosis assays performed. Activity and localisation of p53 and NFκB was determined after co-treatment of cells.ResultsPre-treatment of cervical cancer cells with INI-43 at sublethal concentrations enhanced cisplatin sensitivity, evident through decreased cell viability and enhanced apoptosis. Kpnβ1 knock-down cells similarly displayed increased sensitivity to cisplatin. Combination index determination using the Chou-Talalay method revealed that INI-43 and cisplatin engaged in synergistic interactions. p53 was found to be involved in the cell death response to combination treatment as its inhibition abolished the enhanced cell death observed. INI-43 pre-treatment resulted in moderately stabilized p53 and induced p53 reporter activity, which translated to increased p21 and decreased Mcl-1 upon cisplatin combination treatment. Furthermore, cisplatin treatment led to nuclear import of NFκB, which was diminished upon pre-treatment with INI-43. NFκB reporter activity and expression of NFκB transcriptional targets, cyclin D1, c-Myc and XIAP, showed decreased levels after combination treatment compared to single cisplatin treatment and this associated with enhanced DNA damage.ConclusionsTaken together, this study shows that INI-43 pre-treatment significantly enhances cisplatin sensitivity in cervical cancer cells, mediated through stabilization of p53 and decreased nuclear import of NFκB. Hence this study suggests the possible synergistic use of nuclear import inhibition and cisplatin to treat cervical cancer.

Mutations in Novel COVID-19 Make it More Dangerous: Prevention Via Scientific Approaches

We computed several genomes of COVID-19 information from GISAID, NCBI, and NMDC. Sequence alignment with the strain Bat CoV RaTG13 applied by MAFFT. Genome variable zones of sequence alignment applied the noisy (http://www.bioinf.uni-leipzig.de/Software/noisy/). The protein sequences were gained from NCBI web sites, and the proteins of COVID-19, such as protein sequences, were used to analyze the conserved domain. Several proteins were used for constructing 3-D compounds through homology modeling. Moreover, we show that N-terminal deletions of karyopherin alpha 2 that no longer linkage to karyopherin beta 1 retain ORF6 binding activity but no longer block STAT1 nuclear enter. Recombinant SARS-CoV lacking ORF6 did not close karyopherin alpha 2 to the ER/Golgi membrane and led to the import of the STAT1 complex into the nucleus. Some genomes of different coronaviruses applying BAST and MAFFT software have been estimated, and other genomes have been chosen. It has been unraveled that COVID-19 can generate a new mutation, especially in glycoproteins.

Genome-wide screening reveals a role for subcellular localization of CRBN in the anti-myeloma activity of pomalidomide

Pomalidomide, a derivative of thalidomide, is an effective treatment for multiple myeloma. The drug exerts its effects through CRBN, a component of the E3 ubiquitin ligase complex CRL4CRBN. To search for novel factors involved in the anti-cancer activity of pomalidomide, we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity. Genes encoding components of the ubiquitin-proteasome pathway, such as subunits of the CRL4CRBN complex, the COP9 signalosome, and the 26S proteasome, were among the pomalidomide-affecting genes. Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene. KPNB1 was required for the nuclear import of CRBN and for the CRBN-directed, pomalidomide-dependent degradation of a clinically relevant substrate, the transcription factor Aiolos. By contrast, the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm, indicating that subcellular distribution of CRBN is critical for the efficacy of thalidomide-based medications.

Inhibition of Karyopherin beta 1 suppresses prostate cancer growth.

Prostate cancer (PCa) initiation and progression requires activation of numerous oncogenic signaling pathways. Nuclear-cytoplasmic transport of oncogenic factors is mediated by Karyopherin proteins during cell transformation. However, the role of nuclear transporter proteins in PCa progression has not been well defined. Here, we report that the KPNB1, a key member of Karyopherin beta subunits, is highly expressed in advanced prostate cancers. Further study showed that targeting KPNB1 suppressed the proliferation of prostate cancer cells. The knockdown of KPNB1 reduced nuclear translocation of c-Myc, the expression of downstream cell cycle modulators, and phosphorylation of regulator of chromatin condensation 1 (RCC1), a key protein for spindle assembly during mitosis. Meanwhile, CHIP assay demonstrated the binding of c-Myc to KPNB1 promoter region, which indicated a positive feedback regulation of KPNB1 expression mediated by the c-Myc. In addition, NF-κB subunit p50 translocation to nuclei was blocked by KPNB1 inhibition, which led to an increase in apoptosis and a decrease in tumor sphere formation of PCa cells. Furthermore, subcutaneous xenograft tumor models with a stable knockdown of KPNB1 in C42B PCa cells validated that the inhibition of KPNB1 could suppress the growth of prostate tumor in vivo. Moreover, the intravenously administration of importazole, a specific inhibitor for KPNB1, effectively reduced PCa tumor size and weight in mice inoculated with PC3 PCa cells. In summary, our data established the functional link between KPNB1 and PCa prone c-Myc, NF-kB, and cell cycle modulators. More importantly, inhibition of KPNB1 could be a new therapeutic target for PCa treatment.

60P - Investigating the role of nuclear sphingosine kinase 1 (SphK1) in lung cancer

Background: Sphingosine kinases (SphKs) are enzymes catalyzing the formation of a bioactive lipid messenger, sphingosine-1-phosphate (S1P) through phosphorylation of sphingosine. So far, two isoforms of SphK have been described: SphK1, which has cytosolic distribution, and SphK2, known to locate predominantly in the nucleus. Overactivation of SphKs is involved in tumor growth and progression through regulation of cell proliferation and apoptosis. Methods: We studied the expression, the intracellular localization and the activity of SphK1 and SphK2 in non-small cell lung cancer (NSCLC) cell lines and in cancer patients. Results: In NSCLC cells, Sphk1 was found in the cytosol, while Sphk2 showed a nuclear localization. Surprisingly, we observed SphK1 expression also in the nuclear compartment. In several of the tested cell lines, the expression of SphK1 was even higher in the nucleus compared to the cytosol. Consistently, in a NSCLC tissue microarray, SphK1 nuclear expression was detected in a wide number of samples. Based on these data, we hypothesize the presence of a nuclear import signal in the SphK1 sequence, probably recognized by Karyopherin beta2. We found, in the aminoacidic sequence of SphK1, a R/K/H-X(5)-P-Y C-terminal motif within a structurally disordered and positively charged regions of about 30 amino acids. In order to dissect the role of nuclear SphK1, we used different tools able to modulate nuclear import/export. Moreover, specific inhibitors of SphKs were tested. Among them, fingolimod (FTY720), an oral drug initially defined as a S1P receptors antagonist but also acting as a SphK1 inhibitor, is effective in NSCLC cell lines: a reduction of cell density of about 50% is obtained with a drug concentration of about 2.5 µM. Interestingly, fingolimod seems to be more effective in cells with a nuclear/cytosolic ratio of SphK1 expression > 1. Conclusions: We demonstrate for the first time that SphK1 has a nuclear localization in NSCLC and we are now investigating its biological role in this context. Moreover, we propose nuclear SphK1 as a novel druggable target in human NSCLC cells. Since fingolimod is a clinically available drug, our results may suggest SphK1 targeting as a therapeutic opportunity to be tested in NSCLC patients. Legal entity responsible for the study: Prof. Roberto Bianco Funding: Has not received any funding Disclosure: All authors have declared no conflicts of interest.

Decreased KPNB1 Expression is Induced by PLK1 Inhibition and Leads to Apoptosis in Lung Adenocarcinoma.

Lung cancer is a major cause of death worldwide, with lung adenocarcinoma being the most frequently diagnosed subtype in Japan. Finding the target of an anticancer drug can improve lung adenocarcinoma treatments. Polo-like kinase 1 (PLK1) is an essential mitotic kinase in mitotic progression, and PLK1 inhibition induces cell cycle arrest and apoptosis in tumor cells. In addition, a variety of PLK1 inhibitors have been identified for cancer treatments. In this study, we looked for the target gene of the anticancer drug that has synergy with PLK1 inhibitors. We identified karyopherin beta 1 (KPNB1) as a possible target for lung adenocarcinoma treatment. We found that PLK1 inhibition decreased KPNB1 expression in lung adenocarcinoma cells and KPNB1 depletion inhibited cell proliferation via apoptosis. The same apoptosis signaling pathway may be activated because the expression of common apoptosis-related genes was decreased by PLK1 and KPNB1 silencing; however, the time course of cell growth inhibition was somewhat different. Cell cycle analysis showed that KPNB1 depletion increased the proportion of cells at the G0/G1 phase, although cells also accumulated at the G2/M phase in PLK1-depleted cells. Our findings suggest that decreased KPNB1 expression may be associated with the apoptosis induced by PLK1 inhibition.

KPNA3-knockdown eliminates the second heat shock protein peak associated with the heat shock response of male silkworm pupae (Bombyx mori) by reducing heat shock factor transport into the nucleus

In this study, we investigated the role of karyopherin alpha 3 in the heat shock response in male silkworm pupae. Karyopherin alpha recognizes the classical nuclear location sequence on proteins and transports them into the nucleus by forming a trimetric complex with karyopherin beta. Three predicted karyopherin alphas (KPNA1, KPNA2 and KPNA3) have been identified from the silkworm Bombyx mori. Pull-down assay result showed that KPNA3 can pull down heat shock transcription factor (HSF) from proteins extracted from tissues using non-denature lysis buffer. After 45°C heat shock on male B. mori pupae for 30min, we identified two heat shock protein (HSP) mRNA expression peaks correlating with HSP19.9, HSP20.4 and HSP25.4 at 4h (peak 1) and 24h (peak 2). The second peak was eliminated after knockdown of KPNA3. Similar results were obtained following knockdown of HSF, which is the trans-activating factor of heat shock. However, KPNA3 knockdown was not accompanied by the decreased HSF protein levels at 24h after heat shock which were observed following HSF knockdown. We also expressed recombinant protein GST-KPNA3 and His-HSF in Escherichia coli to perform GST pull-down assay and the result confirmed the interaction between KPNA3 and HSF. We concluded that KPNA3 knockdown eliminates the second heat shock protein peak in the heat shock response of male silkworm pupae by reducing HSF transport into the nucleus.

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif.

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (774gpgfgssppGaeaapslRyvPY795) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X 2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.

KPNB1 (karyopherin (importin) beta 1)

Review on KPNB1 (karyopherin (importin) beta 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract 5118: Cysteine (C)-X-C receptor 4 undergoes transportin 1 dependent nuclear localization and is functional at the nucleus of metastatic prostate cancer cells.

Abstract The G-Protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4) plays an important role in prostate cancer (PCa) metastasis. To date, considerable evidence support the presence of GPCRs at the plasma membrane (PM), or within the perinuclear/nuclear compartments of cells and have been suggested to regulate a number of physiological processes including cell proliferation, survival, inflammatory responses and tumorigenesis. In accordance with previous clinical observations in diverse tumor tissues, we observed ubiquitous expression of CXCR4 in metastatic PCa tissues and several PCa cells lines. Additionally, Gαi immunoprecipitation and calcium mobilization studies elucidated that nuclear CXCR4 was functional and participated in G-protein signaling. However, a mechanism by which CXCR4 is translocated to the nucleus is poorly understood.An initial search of the CXCR4 sequence via the PSORT II NLS prediction software program revealed a putative, nontraditional nuclear localization sequence (NLS), “RPRK” in CXCR4.Additionally,the members of karyopherin beta (β) family, Karyopherin-β2 or Transportin 1 (TRN1) were reported to be involved in transportation of various cargos into the nucleus, including the chemokine GPCRs. Collectively, based on our results and previous studies, we suggest that the NLS, “RPRK”and TRN1 play a role in translocation of CXCR4 into the nucleus of advanced PCa. To determine the involvement of “RPRK," manipulations of the NLS revealed that single point mutations of basic resides within the NLS was insufficient to eliminate to abrogate nuclear translocation of CXCR4. However, when the NLS was deleted, nuclear CXCR4 was not detected. Additionally, TRN1 immunoprecipitation and siRNA-TRN1 studies delineated an association between CXCR4 and TRN1,and that nuclear translocation of CXCR4 depended on TRN1. Little information is available about nuclear GPCRs and nuclear CXCR4 in cancer. Thus, the significance of the study is that since CXCR4 is a major signaling receptor involved in PCa metastasis, the dualistic localization pattern and function at the nucleus may encourage recurrence,metastasis and poorer prognosis after tumors have been treated with therapy that targets CXCR4.In conclusion, our studies will provide insight to the development of therapeutics towards CXCR4 and transport mechanisms, as current therapy targets membrane receptors, which may be ineffective if CXCR4 is translocated and functional inside of the cell. Citation Format: Ayesha S. Don-Salu-Hewage, Siu Y. Chan, Kathleen McAndrews, Mahandranauth A. Chetram, Michelle R. Dawson, Cimona V. Hinton. Cysteine (C)-X-C receptor 4 undergoes transportin 1 dependent nuclear localization and is functional at the nucleus of metastatic prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2013-5118

A 3D Physical Model of Karyopherin‐β2

Karyopherin beta 2 (Kapβ2) is an importin that transports various cargo proteins into the nucleus through interactions with nucleoporins, which are proteins of the nuclear pore complex (NPC). One might overlook the significance of this protein but it actually plays a crucial role in the human body. In fact Kapβ2 is responsible for mediating transport of RNA‐binding proteins involved in RNA translation. Consequently, without this protein, we would not be able to exist. The structure of Kapβ2 is composed of 20 antiparallel helices called HEAT repeats. These HEAT repeats contribute to Kapβ2's large superhelical shape. The protein is shown to form two arches: one at the N‐terminal and the other at the C‐terminal. Through recognition of a nuclear localization signal (NLS) located on its cargo, Kapβ2 binds to its cargo via its C‐terminal arch. Release of the cargo is mediated by RanGTP, which once bound, modifies the shape of Kapβ2. This slight conformational change results in the dissociation of the substrate from its binding site. The Hostos Lincoln Academy of Science SMART (Students Modeling a Research Topic) Team and MSOE have designed and made a physical model by three‐dimensional (3‐ D) printing technology, revealing the substrate and RanGTP binding domain. Supported by grants from the HHMI Pre‐ College Program and the Camille and Henry Dreyfus Foundation.

Proteome analysis in adipose tissue of ob/ob mice in response to chitosan oligosaccharides treatment

Adipose tissue plays a central role in the development of obesity, and thus characterization of the molecular changes related to obesity in this tissue is a main concern. Recently we identified chitosan oligosaccharides (CO) as a potent adipogenic inhibitor in 3T3-L1 cells. In the current study, a proteomic approach was used to investigate the anti-obesity effect of CO in white adipose tissue of ob/ob mice. CO administration significantly lowered body weight gain and epididymal WAT mass compared to control animals. In addition, twenty-five proteins were found to be differentially expressed between the two groups of animals in response to CO treatment. Expression changes in Karyopherin beta 1, indoleamine-pyrrole 2,3-dioxygenase, and retinoic acid binding protein were associated here for the first time with obesity. Immunoblotting studies of adipocyte protein 2 (aP2) and aquaporin-7 also showed amelioration in their levels in WAT. Furthermore, the results of adipose tissue specific gene expressions of aP2, adiponectin, TNF-α, and IL-6 were in good agreement with improved levels of obesity. Gene expression of PPARg and SREBP-1c were also down-regulated by CO treatment. The results suggest that the anti-obesity effect of CO might be mediated by the modulation of adipokines and adipose tissue specific genes.

Evolutionary and Transcriptional Analysis of Karyopherin β Superfamily Proteins

In eukaryotes, karyopherin beta superfamily proteins mediate nucleocytoplasmic transport of macromolecules. We investigated the evolutionary and transcriptional patterns of these proteins using bioinformatics approaches. No obvious homologs were found in prokaryotes, but an extensive set of beta-karyopherin proteins was found in yeast. Among 14 beta-karyopherins of Saccharomyces cerevisiae, eight corresponded to their human orthologs directly without diversification, two were lost, and the remaining four proteins exhibited gene duplications by different mechanisms. We also identified beta-karyopherin orthologs in Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Gallus gallus, and Mus musculus. beta-Karyopherins were ubiquitously but nonuniformly expressed in distinct cells and tissues. In yeast and mice, the titer of some beta-karyopherin transcripts appeared to be regulated both during the cell cycle and during development. Further virtual analysis of promoter binding elements suggested that the transcription factors SP1, NRF-2, HEN-1, RREB-1, and nuclear factor Y regulate expression of most beta-karyopherin genes. These findings emphasize new mechanisms in functional diversification of beta-karyopherins and regulation of nucleocytoplasmic transport.

Severe Acute Respiratory Syndrome Coronavirus ORF6 Antagonizes STAT1 Function by Sequestering Nuclear Import Factors on the Rough Endoplasmic Reticulum/Golgi Membrane

The host innate immune response is an important deterrent of severe viral infection in humans and animals. Nuclear import factors function as key gatekeepers that regulate the transport of innate immune regulatory cargo to the nucleus of cells to activate the antiviral response. Using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model, we demonstrate that SARS-COV ORF6 protein is localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, where it binds to and disrupts nuclear import complex formation by tethering karyopherin alpha 2 and karyopherin beta 1 to the membrane. Retention of import factors at the ER/Golgi membrane leads to a loss of STAT1 transport into the nucleus in response to interferon signaling, thus blocking the expression of STAT1-activated genes that establish an antiviral state. We mapped the region of ORF6, which binds karyopherin alpha 2, to the C terminus of ORF6 and show that mutations in the C terminus no longer bind karyopherin alpha 2 or block the nuclear import of STAT1. We also show that N-terminal deletions of karyopherin alpha 2 that no longer bind to karyopherin beta 1 still retain ORF6 binding activity but no longer block STAT1 nuclear import. Recombinant SARS-CoV lacking ORF6 did not tether karyopherin alpha 2 to the ER/Golgi membrane and allowed the import of the STAT1 complex into the nucleus. We discuss the likely implications of these data on SARS-CoV replication and pathogenesis.

Traffic Rules for nuclear import pathways: Karyopherin beta2 and Kap104p

Proteomic studies show 40% of yeast proteins enter the nucleus. By analogy, >10,000 human proteins will enter the nucleus. This huge protein traffic is mediated by 10 different import-Kapbeta2 (Importin-betas), each selectively binding a set of substrates by recognizing specific NLSs. Structural and biochemical analyses of Kapbeta2/Transportin bound to the NLS of hnRNP A1 have led to formulation of 3 rules for NLS recognition by Kapbeta2: NLSs are structurally disordered in free substrates, have overall basic character, and possess a central hydrophobic or basic motif followed by a R/H/KX(2–5)PY consensus sequence. These rules identified NLSs in 7 previously known Kapbeta2 substrates and bioinformatics analysis uncovered 81 new candidate substrates. These studies define and validate a new NLS termed the PY-NLS, which could not be predicted by primary sequence analysis alone. PY-NLSs are divided into hydrophobic or hPY-NLSs and basic or bPY-NLSs based on their diverse central motifs. In the structure of Kapbeta2 bound to the bPY-NLS of hnRNP M, three-dimensional coincidence of the central basic/hydrophobic and the C-terminal R/H/KX(2–5)PY motifs provide structural evidence for their importance, conservation and thus their designation as consensus sequences. Comparative structural analysis also reveals the origin of recognition of diverse PY-NLS sequences. Furthermore, structural and thermodynamic analyses of Kapbeta2-NLS interactions have led to the design of a pathway-selective nuclear import inhibitor. Finally, we have validated and refined the human Kapbeta2 NLS rules for the orthologous Kap104p pathway in S. cerevisiae where the rules have identified >100 new Kap104 ligands. This near-complete traffic map of a single nuclear import pathway in yeast allows for bioinformatics analysis to explore potential functional linkages in nuclear trafficking.

Identification of karyopherin β as an immunogenic antigen of the malaria parasite using immune mice and human sera

A differential immunoscreening of the lambdagt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent-phase mouse sera) and immune sera collected from healthy adults, to identify novel cross-reactive and possibly protective antigens of the parasite. One clone, with an insert size of 1132 bp that reacted strongly with both the sera was selected. The insert was found to be a part of the P. falciparum karyopherin beta (PfKbeta) homologue. RT-PCR and Northern blot analysis confirmed the expression of PfKbeta in the blood stages of the parasite. The approximately 110 kDa protein was localized in the cytoplasm at the ring and trophozoite, and in the parasitophorous vacuole at the schizont stage. Two large fragments of PfKbeta representing the N- and C-terminal halves were expressed in E. coli. The recombinant proteins were highly immunogenic in mice, and also found to be the target for immune response in natural infections of Plasmodium spp. Anti-sera against the protein showed a low level of anti-parasitic activity. Immunization with recombinant PfKbeta fragments was only partially protective against a heterologous challenge infection in mice. Our results show that the parasite releases a highly immunogenic, cytoplasmic protein into the host which may not contribute to the development of protective immunity.

A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.