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Anne Kallioniemi - One of the best experts on this subject based on the ideXlab platform.
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Correction to: Depletion of nuclear import protein Karyopherin Alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells.
BMC cancer, 2019Co-Authors: Elisa M. Vuorinen, Nina K. Rajala, Teemu O. Ihalainen, Anne KallioniemiAbstract:Following publication of the original article [1], the authors notified us that the Additional File 1 contains reviewer comments instead of the Supplementary tables.
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Depletion of nuclear import protein Karyopherin Alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells.
BMC cancer, 2018Co-Authors: Elisa M. Vuorinen, Teemu O. Ihalainen, Nina Rajala, Anne KallioniemiAbstract:Following publication of the original article [1], the authors notified us that the Additional File 1 contains reviewer comments instead of the Supplementary tables.
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Depletion of nuclear import protein Karyopherin Alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells
BMC, 2018Co-Authors: Elisa M. Vuorinen, Nina K. Rajala, Teemu O. Ihalainen, Anne KallioniemiAbstract:Abstract Background Nucleocytoplasmic transport is a tightly regulated process carried out by specific transport machinery, the defects of which may lead to a number of diseases including cancer. Karyopherin Alpha 7 (KPNA7), the newest member of the Karyopherin Alpha nuclear importer family, is expressed at a high level during embryogenesis, reduced to very low or absent levels in most adult tissues but re-expressed in cancer cells. Methods We used siRNA-based knock-down of KPNA7 in cancer cell lines, followed by functional assays (proliferation and cell cycle) and immunofluorescent stainings to determine the role of KPNA7 in regulation of cancer cell growth, proper mitosis and nuclear morphology. Results In the present study, we show that the silencing of KPNA7 results in a dramatic reduction in pancreatic and breast cancer cell growth, irrespective of the endogenous KPNA7 expression level. This growth inhibition is accompanied by a decrease in the fraction of S-phase cells as well as aberrant number of centrosomes and severe distortion of the mitotic spindles. In addition, KPNA7 depletion leads to reorganization of lamin A/C and B1, the main nuclear lamina proteins, and drastic alterations in nuclear morphology with lobulated and elongated nuclei. Conclusions Taken together, our data provide new important evidence on the contribution of KPNA7 to the regulation of cancer cell growth and the maintenance of nuclear envelope environment, and thus deepens our understanding on the impact of nuclear transfer proteins in cancer pathogenesis
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Abstract LB-272: KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology
Molecular and Cellular Biology Genetics, 2017Co-Authors: Elisa M. Vuorinen, Teemu O. Ihalainen, Hanna E. Rauhala, Nina Rajala, Anssi Nurminen, Vesa P Hytonen, Anne KallioniemiAbstract:Background: Bidirectional nucleocytoplasmic transport unique for eukaryotic cells is a tightly regulated process carried out by specific transport machinery. Its defects result in incorrect localization of proteins that might subsequently lead to diversity of diseases including cancer. Karyopherin Alpha 7 (KPNA7) is the newest Karyopherin Alpha nuclear importer family member, mainly expressed in early embryogenesis and oocytes. However, its expression is reactivated in some cancer cells. We previously showed that KPNA7 promotes the malignant properties of pancreatic cancer cells overexpressing the gene. Here, we identified KPNA7 cargo proteins and further explored the functional consequences of KPNA7 expression for cell proliferation, cell cycle distributions and maintenance of proper nuclear morphology in a panel of pancreatic and breast cancer cell lines with varying KPNA7 expression levels. Methods: To isolate KPNA7 cargo proteins, an affinity-based protein pull-down was performed from stable, tagged KPNA7-overexpressing pancreatic cancer cell lines and the KPNA7 binding partners were identified with mass spectrometry. For functional studies, KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers and cell cycle distributions were determined 72 to 96 h after transfection and compared to those in corresponding controls. To study mitotic spindle assembly and nuclear morphology, immunofluorescent labeling of γ-tubulin and different nuclear envelope proteins was performed 96 h after transfections. Results: The protein pull-down and mass spectrometry yielded multiple proteins co-purifying with KPNA7. Two cargos, zinc finger protein 414 and major vault protein were successfully validated to bind KPNA7 in vitro and shown to be transported into the nucleus by KPNA7. Additionally, these cargos were shown to possess growth regulatory roles in pancreatic cancer cells. The silencing of KPNA7 in six pancreatic and breast cell lines decreased cell proliferation in all cell lines expressing the gene, whereas both cancer and normal cell lines without endogenous KPNA7 expression were not affected. Also, a decrease in the fraction of proliferating S-phase cells was detected. KPNA7 depletion led to aberrant mitotic spindle assembly and abnormal number of centrosomes. Interestingly, a reorganization of nuclear envelope proteins was observed, resulting in distinct change from round to lobular nuclear morphology. Conclusions: The present results indicate KPNA7 as a central regulator of cancer cell growth and cell cycle, and identify KPNA7 cargo proteins participating in this regulation. We also show that KPNA7, probably via its cargos, has roles beyond nuclear transfer. Our results suggest that KPNA7 functions in the regulation of proper mitosis and organization of the mitotic spindle and acts in the maintenance of nuclear envelope structure and nuclear morphology. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Teemu Ihalainen, Hanna E. Rauhala, Anssi Nurminen, Vesa P. Hytonen, Anne Kallioniemi. KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2017-LB-272
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search for kpna7 cargo proteins in human cells reveals mvp and znf414 as novel regulators of cancer cell growth
Biochimica et Biophysica Acta, 2017Co-Authors: Elisa M. Vuorinen, Hanna E. Rauhala, Nina Rajala, Anssi Nurminen, Vesa P Hytonen, Anne KallioniemiAbstract:Abstract Karyopherin Alpha 7 (KPNA7) belongs to a family of nuclear import proteins that recognize and bind nuclear localization signals (NLSs) in proteins to be transported to the nucleus. Previously we found that KPNA7 is overexpressed in a subset of pancreatic cancer cell lines and acts as a critical regulator of growth in these cells. This characteristic of KPNA7 is likely to be mediated by its cargo proteins that are still mainly unknown. Here, we used protein affinity chromatography in Hs700T and MIA PaCa-2 pancreatic cancer cell lines and identified 377 putative KPNA7 cargo proteins, most of which were known or predicted to localize to the nucleus. The interaction was confirmed for two of the candidates, MVP and ZNF414, using co-immunoprecipitation, and their transport to the nucleus was hindered by siRNA based KPNA7 silencing. Most importantly, silencing of MVP and ZNF414 resulted in marked reduction in Hs700T cell growth. In conclusion, these data uncover two previously unknown human KPNA7 cargo proteins with distinct roles as novel regulators of pancreatic cancer cell growth, thus deepening our understanding on the contribution of nuclear transport in cancer pathogenesis.
Christopher F. Basler - One of the best experts on this subject based on the ideXlab platform.
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VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability
Journal of virology, 2017Co-Authors: Toni M. Schwarz, Megan R. Edwards, Audrey Diederichs, Joshua B. Alinger, Daisy W. Leung, Gaya K. Amarasinghe, Christopher F. BaslerAbstract:Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of Karyopherin Alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCE The interaction of Ebola virus (EBOV) VP24 protein with host Karyopherin Alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.
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Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells.
Virology, 2015Co-Authors: Alicia R Feagins, Christopher F. BaslerAbstract:Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-Alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, and PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to Karyopherin Alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV.
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Ebolavirus VP24 Binding to Karyopherins Is Required for Inhibition of Interferon Signaling
Journal of Virology, 2009Co-Authors: Mathieu Mateo, Christopher F. Basler, St. Patrick Reid, Lawrence W. Leung, Viktor E. VolchkovAbstract:The Ebolavirus VP24 protein counteracts Alpha/beta interferon (IFN-α/β) and IFN-γ signaling by blocking the nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1). According to the proposed model, VP24 binding to members of the NPI-1 subfamily of Karyopherin Alpha (KPNα) nuclear localization signal receptors prevents their binding to PY-STAT1, thereby preventing PY-STAT1 nuclear accumulation. This study now identifies two domains of VP24 required for inhibition of IFN-β-induced gene expression and PY-STAT1 nuclear accumulation. We demonstrate that loss of function correlates with loss of binding to KPNα proteins. Thus, the VP24 IFN antagonist function requires the ability of VP24 to interact with KPNα.
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NOTES Ebolavirus VP24 Binding to Karyopherins Is Required for Inhibition of Interferon Signaling
2009Co-Authors: Mathieu Mateo, Christopher F. Basler, Lawrence W. Leung, St. Patrick Reid, Viktor E. VolchkovAbstract:The Ebolavirus VP24 protein counteracts Alpha/beta interferon (IFN-/) and IFN- signaling by blocking the nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1). According to the proposed model, VP24 binding to members of the NPI-1 subfamily of Karyopherin Alpha (KPN) nuclear localization signal receptors prevents their binding to PY-STAT1, thereby preventing PY-STAT1 nuclear accumulation. This study now identifies two domains of VP24 required for inhibition of IFN--induced gene expression and PY-STAT1 nuclear accumulation. We demonstrate that loss of function correlates with loss of binding to KPN proteins. Thus, the VP24 IFN antagonist function requires the ability of VP24 to interact with KPN. In the order Mononegavirales, Ebolavirus and Marburgvirus belong to the family Filoviridae (12). Ebolaviruses are respon-sible for outbreaks in central Africa of severe hemorrhagic fever in humans and nonhuman primates, with human fatality rates of up to 90 % (16). Currently, there are no licensed vaccines or approved treatments available for filovirus infec-tions. Ebolavirus counteracts synthesis of Alpha/beta interferon (IFN-/) and cellular responses to IFN-// (7, 8, 10, 11
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Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily Karyopherin Alpha proteins with activated STAT1
2007Co-Authors: St. Patrick Reid, Charalampos Valmas, Osvaldo Martinez, Freddy Mauricio Sanchez, Christopher F. BaslerAbstract:The Zaire ebolavirus protein VP24 was previously demonstrated to inhibit Alpha/beta interferon (IFN-/)-and IFN--induced nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1) and to inhibit IFN-/- and IFN--induced gene expression. These properties correlated with the ability of VP24 to interact with the nuclear localization signal receptor for PY-STAT1, Karyopherin 1. Here, VP24 is demonstrated to interact not only with overexpressed but also with endogenous Karyopherin 1. Mutational analysis demon-strated that VP24 binds within the PY-STAT1 binding region located in the C terminus of Karyopherin 1. In addition, VP24 was found to inhibit PY-STAT1 binding to both overexpressed and endogenous Karyopherin 1. We assessed the binding of both PY-STAT1 and the VP24 proteins from Zaire, mouse-adapted Zaire, and Reston Ebola viruses for interaction with all six members of the human Karyopherin family. We found, in contrast to previous studies, that PY-STAT1 can interact not only with Karyopherin 1 but also with karyo-pherins 5 and 6, which together comprise the NPI-1 subfamily of Karyopherin s. Similarly, all three VP24s bound and inhibited PY-STAT1 interaction with Karyopherins 1, 5, and 6. Consistent with their ability to inhibit the Karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN--induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with Karyopherins 1, 5, or 6 by binding withi
Elisa M. Vuorinen - One of the best experts on this subject based on the ideXlab platform.
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Correction to: Depletion of nuclear import protein Karyopherin Alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells.
BMC cancer, 2019Co-Authors: Elisa M. Vuorinen, Nina K. Rajala, Teemu O. Ihalainen, Anne KallioniemiAbstract:Following publication of the original article [1], the authors notified us that the Additional File 1 contains reviewer comments instead of the Supplementary tables.
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Depletion of nuclear import protein Karyopherin Alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells.
BMC cancer, 2018Co-Authors: Elisa M. Vuorinen, Teemu O. Ihalainen, Nina Rajala, Anne KallioniemiAbstract:Following publication of the original article [1], the authors notified us that the Additional File 1 contains reviewer comments instead of the Supplementary tables.
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Depletion of nuclear import protein Karyopherin Alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells
BMC, 2018Co-Authors: Elisa M. Vuorinen, Nina K. Rajala, Teemu O. Ihalainen, Anne KallioniemiAbstract:Abstract Background Nucleocytoplasmic transport is a tightly regulated process carried out by specific transport machinery, the defects of which may lead to a number of diseases including cancer. Karyopherin Alpha 7 (KPNA7), the newest member of the Karyopherin Alpha nuclear importer family, is expressed at a high level during embryogenesis, reduced to very low or absent levels in most adult tissues but re-expressed in cancer cells. Methods We used siRNA-based knock-down of KPNA7 in cancer cell lines, followed by functional assays (proliferation and cell cycle) and immunofluorescent stainings to determine the role of KPNA7 in regulation of cancer cell growth, proper mitosis and nuclear morphology. Results In the present study, we show that the silencing of KPNA7 results in a dramatic reduction in pancreatic and breast cancer cell growth, irrespective of the endogenous KPNA7 expression level. This growth inhibition is accompanied by a decrease in the fraction of S-phase cells as well as aberrant number of centrosomes and severe distortion of the mitotic spindles. In addition, KPNA7 depletion leads to reorganization of lamin A/C and B1, the main nuclear lamina proteins, and drastic alterations in nuclear morphology with lobulated and elongated nuclei. Conclusions Taken together, our data provide new important evidence on the contribution of KPNA7 to the regulation of cancer cell growth and the maintenance of nuclear envelope environment, and thus deepens our understanding on the impact of nuclear transfer proteins in cancer pathogenesis
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Abstract LB-272: KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology
Molecular and Cellular Biology Genetics, 2017Co-Authors: Elisa M. Vuorinen, Teemu O. Ihalainen, Hanna E. Rauhala, Nina Rajala, Anssi Nurminen, Vesa P Hytonen, Anne KallioniemiAbstract:Background: Bidirectional nucleocytoplasmic transport unique for eukaryotic cells is a tightly regulated process carried out by specific transport machinery. Its defects result in incorrect localization of proteins that might subsequently lead to diversity of diseases including cancer. Karyopherin Alpha 7 (KPNA7) is the newest Karyopherin Alpha nuclear importer family member, mainly expressed in early embryogenesis and oocytes. However, its expression is reactivated in some cancer cells. We previously showed that KPNA7 promotes the malignant properties of pancreatic cancer cells overexpressing the gene. Here, we identified KPNA7 cargo proteins and further explored the functional consequences of KPNA7 expression for cell proliferation, cell cycle distributions and maintenance of proper nuclear morphology in a panel of pancreatic and breast cancer cell lines with varying KPNA7 expression levels. Methods: To isolate KPNA7 cargo proteins, an affinity-based protein pull-down was performed from stable, tagged KPNA7-overexpressing pancreatic cancer cell lines and the KPNA7 binding partners were identified with mass spectrometry. For functional studies, KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers and cell cycle distributions were determined 72 to 96 h after transfection and compared to those in corresponding controls. To study mitotic spindle assembly and nuclear morphology, immunofluorescent labeling of γ-tubulin and different nuclear envelope proteins was performed 96 h after transfections. Results: The protein pull-down and mass spectrometry yielded multiple proteins co-purifying with KPNA7. Two cargos, zinc finger protein 414 and major vault protein were successfully validated to bind KPNA7 in vitro and shown to be transported into the nucleus by KPNA7. Additionally, these cargos were shown to possess growth regulatory roles in pancreatic cancer cells. The silencing of KPNA7 in six pancreatic and breast cell lines decreased cell proliferation in all cell lines expressing the gene, whereas both cancer and normal cell lines without endogenous KPNA7 expression were not affected. Also, a decrease in the fraction of proliferating S-phase cells was detected. KPNA7 depletion led to aberrant mitotic spindle assembly and abnormal number of centrosomes. Interestingly, a reorganization of nuclear envelope proteins was observed, resulting in distinct change from round to lobular nuclear morphology. Conclusions: The present results indicate KPNA7 as a central regulator of cancer cell growth and cell cycle, and identify KPNA7 cargo proteins participating in this regulation. We also show that KPNA7, probably via its cargos, has roles beyond nuclear transfer. Our results suggest that KPNA7 functions in the regulation of proper mitosis and organization of the mitotic spindle and acts in the maintenance of nuclear envelope structure and nuclear morphology. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Teemu Ihalainen, Hanna E. Rauhala, Anssi Nurminen, Vesa P. Hytonen, Anne Kallioniemi. KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2017-LB-272
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search for kpna7 cargo proteins in human cells reveals mvp and znf414 as novel regulators of cancer cell growth
Biochimica et Biophysica Acta, 2017Co-Authors: Elisa M. Vuorinen, Hanna E. Rauhala, Nina Rajala, Anssi Nurminen, Vesa P Hytonen, Anne KallioniemiAbstract:Abstract Karyopherin Alpha 7 (KPNA7) belongs to a family of nuclear import proteins that recognize and bind nuclear localization signals (NLSs) in proteins to be transported to the nucleus. Previously we found that KPNA7 is overexpressed in a subset of pancreatic cancer cell lines and acts as a critical regulator of growth in these cells. This characteristic of KPNA7 is likely to be mediated by its cargo proteins that are still mainly unknown. Here, we used protein affinity chromatography in Hs700T and MIA PaCa-2 pancreatic cancer cell lines and identified 377 putative KPNA7 cargo proteins, most of which were known or predicted to localize to the nucleus. The interaction was confirmed for two of the candidates, MVP and ZNF414, using co-immunoprecipitation, and their transport to the nucleus was hindered by siRNA based KPNA7 silencing. Most importantly, silencing of MVP and ZNF414 resulted in marked reduction in Hs700T cell growth. In conclusion, these data uncover two previously unknown human KPNA7 cargo proteins with distinct roles as novel regulators of pancreatic cancer cell growth, thus deepening our understanding on the contribution of nuclear transport in cancer pathogenesis.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor Karyopherin Alpha.
Cell, 1998Co-Authors: Elena Conti, Gunter Blobel, L Leighton, John KuriyanAbstract:Selective nuclear import is mediated by nuclear localization signals (NLSs) and cognate transport factors known as Karyopherins or importins. Karyopherin Alpha recognizes "classical" monopartite and bipartite NLSs. We report the crystal structure of a 50 kDa fragment of the 60 kDa yeast Karyopherin Alpha, in the absence and presence of a monopartite NLS peptide at 2.2 A and 2.8 A resolution, respectively. The structure shows a tandem array of ten armadillo repeats, organized in a right-handed superhelix of helices. Binding of the NLS peptide occurs at two sites within a helical surface groove that is lined by conserved residues. The structure reveals the determinants of NLS specificity and suggests a model for the recognition of bipartite NLSs.
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Disassembly of RanGTP-Karyopherin beta complex, an intermediate in nuclear protein import.
Journal of Biological Chemistry, 1997Co-Authors: Monique Floer, Gunter Blobel, Michael RexachAbstract:We previously showed that RanGTP forms a 1:1 complex with Karyopherin beta that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G. (1996) J. Biol. Chem. 271, 5313-5316) and Karyopherin beta functionally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). Recycling of both factors for another round of function requires dissociation of the RanGTP-Karyopherin beta complex. Here we show using BIAcoreTM, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that Karyopherin beta and RanGTP are recycled efficiently in a reaction that involves Karyopherin Alpha, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that Karyopherin Alpha first releases RanGTP from Karyopherin beta in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed Karyopherin Alphabeta binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind Karyopherin Alphabeta stimulate recycling of Karyopherin beta and Ran in a manner similar to the C terminus of Nup1.
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Critical role of reverse transcriptase in the inhibitory mechanism of CNI-H0294 on HIV-1 nuclear translocation
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Serguei G. Popov, Michael Rexach, Omar K. Haffar, Peter C. Ulrich, Larisa Dubrovsky, May-ann Lee, Sridhar Pennathur, Yousef Al-abed, Peter J. Tonge, Gunter BlobelAbstract:Abstract HIV-1 replication requires the translocation of viral genome into the nucleus of a target cell. We recently reported the synthesis of an arylene bis(methyl ketone) compound (CNI-H0294) that inhibits nuclear targeting of the HIV-1 genome and thus HIV-1 replication in monocyte cultures. Here we demonstrate that CNI-H0294 inhibits nuclear targeting of HIV-1-derived preintegration complexes by inactivating the nuclear localization sequence of the HIV-1 matrix antigen in a reaction that absolutely requires reverse transcriptase. This drug/reverse transcriptase interaction defines the specificity of its antiviral effect and is most likely mediated by the pyrimidine side-chain of CNI-H0294. After binding to reverse transcriptase, the carbonyl groups of CNI-H0294 react with the nuclear localization sequence of matrix antigen and prevent its binding to Karyopherin Alpha, the cellular receptor for nuclear localization sequences that carries proteins into the nucleus. Our results provide a basis for the development of a novel class of compounds that inhibit nuclear translocation and that can, in principle, be modified to target specific infectious agents.
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Nuclear protein import: Ran-GTP dissociates the Karyopherin Alphabeta heterodimer by displacing Alpha from an overlapping binding site on beta
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract The Alpha subunit of the Karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the Karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of Karyopherin beta's binding sites both for Ran-GTP and for Karyopherin Alpha. We discovered that Karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and Karyopherin Alpha bind to overlapping sites on Karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces Karyopherin Alpha and binds to Karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for Karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute Karyopherin Alpha's binding site to Karyopherin beta.
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The binding site of Karyopherin Alpha for Karyopherin beta overlaps with a nuclear localization sequence
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of Karyopherin Alpha. We found that the C-terminal region of Karyopherin Alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds Karyopherin beta. Surprisingly, Karyopherin Alpha also contains an NLS. Thus, Karyopherin Alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of Karyopherin Alpha overlaps with the binding site of Karyopherin Alpha for Karyopherin beta. Hence, binding of Karyopherin beta to Karyopherin Alpha covers the NLS of Karyopherin Alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of Karyopherin Alpha to a heterologous NLS protein in the presence of Karyopherin beta.
Hanna E. Rauhala - One of the best experts on this subject based on the ideXlab platform.
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Abstract LB-272: KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology
Molecular and Cellular Biology Genetics, 2017Co-Authors: Elisa M. Vuorinen, Teemu O. Ihalainen, Hanna E. Rauhala, Nina Rajala, Anssi Nurminen, Vesa P Hytonen, Anne KallioniemiAbstract:Background: Bidirectional nucleocytoplasmic transport unique for eukaryotic cells is a tightly regulated process carried out by specific transport machinery. Its defects result in incorrect localization of proteins that might subsequently lead to diversity of diseases including cancer. Karyopherin Alpha 7 (KPNA7) is the newest Karyopherin Alpha nuclear importer family member, mainly expressed in early embryogenesis and oocytes. However, its expression is reactivated in some cancer cells. We previously showed that KPNA7 promotes the malignant properties of pancreatic cancer cells overexpressing the gene. Here, we identified KPNA7 cargo proteins and further explored the functional consequences of KPNA7 expression for cell proliferation, cell cycle distributions and maintenance of proper nuclear morphology in a panel of pancreatic and breast cancer cell lines with varying KPNA7 expression levels. Methods: To isolate KPNA7 cargo proteins, an affinity-based protein pull-down was performed from stable, tagged KPNA7-overexpressing pancreatic cancer cell lines and the KPNA7 binding partners were identified with mass spectrometry. For functional studies, KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers and cell cycle distributions were determined 72 to 96 h after transfection and compared to those in corresponding controls. To study mitotic spindle assembly and nuclear morphology, immunofluorescent labeling of γ-tubulin and different nuclear envelope proteins was performed 96 h after transfections. Results: The protein pull-down and mass spectrometry yielded multiple proteins co-purifying with KPNA7. Two cargos, zinc finger protein 414 and major vault protein were successfully validated to bind KPNA7 in vitro and shown to be transported into the nucleus by KPNA7. Additionally, these cargos were shown to possess growth regulatory roles in pancreatic cancer cells. The silencing of KPNA7 in six pancreatic and breast cell lines decreased cell proliferation in all cell lines expressing the gene, whereas both cancer and normal cell lines without endogenous KPNA7 expression were not affected. Also, a decrease in the fraction of proliferating S-phase cells was detected. KPNA7 depletion led to aberrant mitotic spindle assembly and abnormal number of centrosomes. Interestingly, a reorganization of nuclear envelope proteins was observed, resulting in distinct change from round to lobular nuclear morphology. Conclusions: The present results indicate KPNA7 as a central regulator of cancer cell growth and cell cycle, and identify KPNA7 cargo proteins participating in this regulation. We also show that KPNA7, probably via its cargos, has roles beyond nuclear transfer. Our results suggest that KPNA7 functions in the regulation of proper mitosis and organization of the mitotic spindle and acts in the maintenance of nuclear envelope structure and nuclear morphology. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Teemu Ihalainen, Hanna E. Rauhala, Anssi Nurminen, Vesa P. Hytonen, Anne Kallioniemi. KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2017-LB-272
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search for kpna7 cargo proteins in human cells reveals mvp and znf414 as novel regulators of cancer cell growth
Biochimica et Biophysica Acta, 2017Co-Authors: Elisa M. Vuorinen, Hanna E. Rauhala, Nina Rajala, Anssi Nurminen, Vesa P Hytonen, Anne KallioniemiAbstract:Abstract Karyopherin Alpha 7 (KPNA7) belongs to a family of nuclear import proteins that recognize and bind nuclear localization signals (NLSs) in proteins to be transported to the nucleus. Previously we found that KPNA7 is overexpressed in a subset of pancreatic cancer cell lines and acts as a critical regulator of growth in these cells. This characteristic of KPNA7 is likely to be mediated by its cargo proteins that are still mainly unknown. Here, we used protein affinity chromatography in Hs700T and MIA PaCa-2 pancreatic cancer cell lines and identified 377 putative KPNA7 cargo proteins, most of which were known or predicted to localize to the nucleus. The interaction was confirmed for two of the candidates, MVP and ZNF414, using co-immunoprecipitation, and their transport to the nucleus was hindered by siRNA based KPNA7 silencing. Most importantly, silencing of MVP and ZNF414 resulted in marked reduction in Hs700T cell growth. In conclusion, these data uncover two previously unknown human KPNA7 cargo proteins with distinct roles as novel regulators of pancreatic cancer cell growth, thus deepening our understanding on the contribution of nuclear transport in cancer pathogenesis.
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Abstract 76: KPNA7 nuclear import protein - a critical regulator of cancer cell growth
Molecular and Cellular Biology Genetics, 2016Co-Authors: Elisa M. Vuorinen, Hanna E. Rauhala, Nina Rajala, Anne KallioniemiAbstract:Background. In eukaryotic cells, the nucleus is separated from the cytosol by the nuclear envelope, thus requiring a specific machinery for the transport of macromolecules between the two cellular compartments. The nuclear import cycle is strictly regulated and its malfunction results in incorrect localization of proteins, ultimately leading to variety of diseases including cancer. Karyopherin Alpha 7 (KPNA7) is the newest member of the Karyopherin Alpha family of nuclear importers. KPNA7 is mainly expressed in early embryogenesis and oocytes as well as in some cancer cells. We previously showed that silencing of KPNA7 in pancreatic cancer cell lines with high endogenous expression dramatically reduced cell proliferation and anchorage-independent growth, through a G1 cell cycle arrest and transcriptional induction of p21 expression. Here, we further explored the functional importance of KPNA7 in a large panel of pancreatic and breast cancer cell lines and identified its cargo proteins that may be responsible for the observed phenotypes. Methods. KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers was determined 72 to 96 h after transfection and compared to those in corresponding controls. To isolate KPNA7 cargo proteins, stable tagged KPNA7-overexpressing pancreatic cancer cell lines were established. An affinity-based protein pull-down was performed and the KPNA7-interacting partners were identified with mass spectrometry. Results. The silencing of KPNA7 in six pancreatic and breast cell lines led to decreased proliferation in all cell lines with low to medium expression level, whereas both cancer and normal cell lines with absolutely no KPNA7 expression were not affected, thus confirming the specificity of the phenotype to KPNA7 silencing. The results were also consistent in breast cancer cell lines, implicating a role not restricted to pancreatic cancer. Interestingly, in some cell lines a distinct change from round to lobular nuclear morphology was observed, suggesting that KPNA7 may also participate in the maintenance of nuclear architecture. The protein pull-down followed by mass spectrometry yielded multiple proteins co-purifying with KPNA7. These included several proteins involved in RNA processing and nucleopore proteins. Many of these were also involved in the regulation of cell cycle and p21 pathway, consistent with the phenotype observed in the functional studies. Conclusions. These results implicate KPNA7 as an important regulator of cancer cell growth and suggest that it might also have other functions in the maintenance of cancer cell homeostasis. We were the first to link KPNA7 to a human malignancy and here we expand that notion beyond pancreatic cancer. We have also identified many putative KPNA7 cargos with functions matching the observed cellular phenotypes. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Hanna E. Rauhala, Anne Kallioniemi. KPNA7 nuclear import protein - a critical regulator of cancer cell growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 76.
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KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro.
Experimental cell research, 2013Co-Authors: Eeva Laurila, Elisa M. Vuorinen, Hanna E. Rauhala, Kimmo Savinainen, Anne KallioniemiAbstract:Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that Karyopherin Alpha 7 (KPNA7), the newest member of the Alpha Karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer.
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Abstract 1753: KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cellsin vitro.
Molecular and Cellular Biology, 2013Co-Authors: Hanna E. Rauhala, Elisa M. Vuorinen, Eeva Laurila, Kimmo Savinainen, Anne KallioniemiAbstract:Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for the early detection of the disease and a shortage of effective therapies. We have previously shown that Karyopherin Alpha 7 (KPNA7) is frequently amplified and overexpressed in pancreatic cancer. KPNA7 is a recently characterized member of the Alpha Karyopherin family of nuclear import receptors. The nuclear import machinery is composed of a complex network of proteins required for the appropriate transport of proteins from the cytoplasm to the nucleus. Abnormal function of this machinery leads to incorrect localization of nuclear proteins and disturbances in cellular homeostasis, which may subsequently contribute to the development of various diseases including cancer. Indeed, previous evidence has linked members of the Karyopherin Alpha family, especially KPNA2, to cancer pathogenesis. In this study we demonstrate that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines as well as in some other cancer types. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a striking reduction in cell growth due to the induction of the cell cycle regulator p21 and subsequent G1 arrest of the cell cycle. In both of these cell lines KPNA7 silencing also resulted in decreased anchorage independent growth, and additionally in the AsPC-1 cells reduced cell migration and invasion. Furthermore, in the Hs700T cells KPNA7 silencing resulted in dramatic morphological change where the cells acquired fibroblast-like shape. This phenotypic change was not explained by induction of EMT or senescence. In conclusion, our data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role of KPNA7 in human cancer. Moreover, the lack of KPNA7 expression in normal adult tissues highlights it as a potential novel therapeutic target for cancer. Further studies are needed to identify the cargo proteins transported by KPNA7. These studies are likely to reveal important new information on nuclear transport and may highlight the molecular mechanisms involved in KPNA7 mediated phenotypes in cancer cells. Citation Format: Hanna E. Rauhala, Eeva Laurila, Elisa Vuorinen, Kimmo Savinainen, Anne Kallioniemi. KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro . [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 1753. doi:10.1158/1538-7445.AM2013-1753
