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A functional study of nucleocytoplasmic transport signals of the EhNCABP166 protein from Entamoeba histolytica

Published online by Cambridge University Press:  23 August 2012

R. URIBE ,
J. ALMARAZ BARRERA MA DE ,
M. ROBLES-FLORES ,
G. MENDOZA HERNÁNDEZ ,
A. GONZÁLEZ-ROBLES ,
R. HERNÁNDEZ-RIVAS ,
N. GUILLEN  and
M. VARGAS
R. URIBE
Affiliation:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F, México Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, D.F, México
J. ALMARAZ BARRERA MA DE
Affiliation:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F, México
M. ROBLES-FLORES
Affiliation:
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F, México
G. MENDOZA HERNÁNDEZ
Affiliation:
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F, México
A. GONZÁLEZ-ROBLES
Affiliation:
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F, México
R. HERNÁNDEZ-RIVAS
Affiliation:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F, México
N. GUILLEN
Affiliation:
Institut Pasteur, Unité Biologie Cellulaire du Parasitisme, Paris, F-75015, France
M. VARGAS*
Affiliation:
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F, México
*
*Corresponding author: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F, México. Tel: +52 55 57473326. Fax: +52 55 5747 3326. E-mail address: [email protected]
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Summary

EhNCABP166 is an Entamoeba histolytica actin-binding protein that localizes to the nucleus and cytoplasm. Bioinformatic analysis of the EhNCABP166 amino acid sequence shows the presence of 3 bipartite nuclear localization signals (NLS) and a nuclear export signal (NES). The present study aimed to investigate the functionality of these signals in 3 ways. First, we fused each potential NLS to a cytoplasmic domain of ehFLN to determine whether the localization of this domain could be altered by the presence of the NLSs. Furthermore, the localization of each domain of EhNCABP166 was determined. Similarly, we generated mutations in the first block of bipartite signals from the domains that contained these signals. Additionally, we added an NES to 2 constructs that were then evaluated. We confirmed the intranuclear localization of EhNCABP166 using transmission electron microscopy. Fusion of each NLS resulted in shuttling of the cytoplasmic domain to the nucleus. With the exception of 2 domains, all of the evaluated domains localized within the nucleus. A mutation in the first block of bipartite signals affected the localization of the domains containing an NLS. The addition of an NES shifted the localization of these domains to the cytoplasm. The results presented here establish EhNCABP166 as a protein containing functional nuclear localization signals and a nuclear export signal.

Keywords

Entamoeba histolyticanuclear localization signal (NLS)nuclear export signal (NES)actin-binding proteinnucleocytoplasmic transport system
Type
Research Article
Information
Parasitology , Volume 139 , Issue 13 , November 2012 , pp. 1697 - 1710
Copyright
Copyright © Cambridge University Press 2012

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References

REFERENCES

Binder, M., Ortner, S., Erben, H., Scheiner, O., Wiedermann, G., Valenta, R. and Duchêne, M. (1995). The basic isoform of profilin in pathogenic Entamoeba histolytica. European Journal of Biochemistry 233, 976–81. http://dx.doi.org/10.1111/j.1432-1033.1995.976_3.x.CrossRefGoogle ScholarPubMed
Blazquez, S., Guigon, G., Weber, C., Syan, S., Sismero, O., Coppée, JY., Labruyère, E. and Guillén, N. (2008). Chemotaxis of Entamoeba histolytica towards the pro-inflammatory cytokine TNF is based on PI3 K signalling, cytoskeleton reorganization and the Galactose/N-acetylgalactosamine lectin activity. Cellular Microbiology 10, 1676–1686. http://dx.doi.org/10.1111/j.1462-5822.2008.01158.x.CrossRefGoogle Scholar
Boulo, S., Akarsu, H., Ruigrok, R. W. and Baudin, F. (2007). Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Research 124, 12–21. http://dx.doi.org/10.1016/j.virusres.2006.09.013.CrossRefGoogle ScholarPubMed
Campos-Parra, A. D., Hernández-Cuevas, N. A., Hernandez-Rivas, R. and Vargas, M. (2010). EhNCABP166: A nucleocytoplasmic actin-binding protein from Entamoeba histolytica. Molecular & Biochemical Parasitology 172, 19–30. http://dx.doi.org/10.1016/j.molbiopara.2010.03.010.CrossRefGoogle ScholarPubMed
Castano, E., Philimonenko, V. V., Kahle, M., Fukalová, J., Kalendová, A., Yildirim, S., Dzijak, R., Dingová-Krásna, H. and Hozák, P. (2010). Actin complexes in the cell nucleus: new stones in an old field. Histochemistry and Cell Biology 133, 607–626. http://dx.doi.org/10.1007/s00418-010-0701-2.CrossRefGoogle Scholar
Chook, Y. M. and Süel, K. E. (2011). Nuclear import by karyopherin-βs: recognition and inhibition. Biochimica et Biophysica Acta 1813, 1593–1606. http://dx.doi.org/10.1016/j.bbamcr.2010.10.014.CrossRefGoogle ScholarPubMed
Coudrier, E., Amblard, F., Zimmer, C., Roux, P., Olivo-Marin, J. C. and Guillén, N. (2005). Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica. Cellular Microbiology 7, 19–27. http://dx.doi.org/10.1111/j.1462-5822.2004.00426.x.CrossRefGoogle ScholarPubMed
Diamond, L. S., Harlow, D. R. and Cunnick, C. C. (1978). A new medium for the axigenic cultivation of Entamoeba histolytica and other Entamoeba. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 431–432.CrossRefGoogle ScholarPubMed
Díaz-Valencia, J. D., Almaraz-Barrera, M. de J., Arias-Romero, L. E., Hernandez-Rivas, R., Rojo-Domínguez, A., Guillén, N. and Vargas, M. (2005). The ABP-120 C-end region from Entamoeba histolytica interacts with sulfatide, a new lipid target. Biochemical and Biophysical Research Communications 338, 1527–1536. http://dx.doi.org/10.1016/j.bbrc.2005.10.119.CrossRefGoogle ScholarPubMed
Díaz-Valencia, J. D., Almaraz-Barrera, M. de J., Jay, D., Hernández-Cuevas, N. A., García, E., González-De la Rosa, C. H., Arias-Romero, L. E., Hernandez-Rivas, R., Rojo-Domínguez, A., Guillén, N. and Vargas, M. (2007). Novel structural and functional findings of the ehFLN protein from Entamoeba histolytica. Cell Motility and the Cytoskeleton 64, 880–896. http://dx.doi.org/10.1002/cm.20232.CrossRefGoogle ScholarPubMed
Ebert, F., Guillén, N., Leippe, M. and Tannich, E. (2000). Molecular cloning and cellular localization of an unusual bipartite Entamoeba histolytica polypeptide with similarity to actin binding proteins. Molecular and Biochemical Parasitology 111, 459–464. http://dx.doi.org/10.1016/S0166-6851(00)00331-5.CrossRefGoogle ScholarPubMed
Fried, H. and Kutay, U. (2003). Nucleocytoplasmic transport: taking an inventory. Cellular and Molecular Life Sciences 60, 1659–1688. doi: 10.1007/s00018-003-3070-3.CrossRefGoogle ScholarPubMed
Görlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annual Review of Cell and Developmental Biology 15, 607–660. http://dx.doi.org/10.1146/annurev.cellbio.15.1.607.CrossRefGoogle ScholarPubMed
Guillén, N. (1996). Role of signaling and cytoskeletal rearrangements in the pathogenesis of Entamoeba histolytica. Trends in Microbiology 4, 191–197. http://dx.doi.org/10.1016/0966-842X(96)10033-0.CrossRefGoogle ScholarPubMed
Güttler, T., Madl, T., Neumann, P., Deichsel, D., Corsini, L., Monecke., T., Ficner, R., Sattler, M. and Görlich, D. (2010). NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nature Structural & Molecular Biology 17, 1367–1376. http://dx.doi.org/10.1038/nsmb.1931.CrossRefGoogle ScholarPubMed
Hamann, L., Nickel, R. and Tannich, E. (1995). Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica. Proceedings of the National Academy of Sciences, USA 92, 8975–8979.CrossRefGoogle ScholarPubMed
Hon, C. C., Nakada-Tsukui, K., Nozaki, T. and Guillén, N. (2010). Dissecting the actin cytoskeleton of Entamoeba histolytica from a genomic perspective. In Anaerobic Parasitic Protozoa, Genomics and Molecular Biology (ed. Graham Clark, C., Johnson, Patricia, J. and Adam, Rodney D.), p. 81–118. Caister Academic Press, Norfolk, UK.Google Scholar
Imamoto, N., Shimamoto, T., Takao, T., Tachibana, T., Kose, S., Matsubae, M., Sekimoto, T., Shimonishi, Y. and Yoneda, Y. (1995). In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import. The EMBO Journal 14, 3617–3626.CrossRefGoogle ScholarPubMed
Kutay, U. and Güttinger, S. (2005). Leucine-rich nuclear-export signals: born to be weak. Trends in Cell Biology 15, 121–124. http://dx.doi.org/10.1016/j.tcb.2005.01.005.CrossRefGoogle ScholarPubMed
Lee, Y. H., Campbell, H. D. and Stallcup, M. R. (2004). Developmentally essential protein flightless I is a nuclear receptor coactivator with actin binding activity. Molecular and Cellular Biology 24, 2103–2117. http://dx.doi.org/10.1128/MCB.24.5.2103-2117.2004.CrossRefGoogle Scholar
Leippe, M. and Herbst, R. (2004). Ancient weapons for attack and defense: the pore forming polypeptides of pathogenic enteric and free-living amoeboid protozoa. The Journal of Eukaryotic Microbiology 51, 516–521. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00286.x.CrossRefGoogle ScholarPubMed
Liu, F., Wagner, S., Campbell, R. B., Nickerson, J. A., Schiffer, C. A. and Ross, A. H. (2005). PTEN enters the nucleus by diffusion. Journal of Cellular Biochemistry 96, 221–234. http://dx.doi.org/10.1002/jcb.20525.CrossRefGoogle ScholarPubMed
Lopez-Girona, A., Furnari, B., Mondesert, O. and Rusell, P. (1999). Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature, London 397, 172–175. http://dx.doi.org/10.1038/16488.CrossRefGoogle Scholar
Magico, A. C. and Bell, J. B. (2011). Identification of a classical bipartite nuclear localization signal in the Drosophila TEA/ATTS protein scalloped. PLoS One 6, e21431. http://dx.plos.org/10.1371/journal.pone.0021431.CrossRefGoogle ScholarPubMed
Malki, S., Boizet-Bonhoure, B. and Poulat, F. (2010). Shuttling of SOX proteins. The International Journal of Biochemistry & Cell Biology 42, 411–416. http://dx.doi.org/10.1016/j.biocel.2009.09.020.CrossRefGoogle ScholarPubMed
Marfori, M., Mynott, A., Ellis, J. J., Mehdi, A. M., Saunders, N. F., Curmi, P. M., Forwood, J. K., Bodén, M. and Kobe, B. (2011). Molecular basis for specificity of nuclear import and prediction of nuclear localization. Biochimica et Biophysica Acta 1813, 1562–1577. http://dx.doi.org/10.1016/j.bbamcr.2010.10.013.CrossRefGoogle ScholarPubMed
Marion, S., Tavares, P., Arhets, P. and Guillén, N. (2004). Signal transduction through the Gal-GalNAc lectin of Entamoeba histolytica involves a spectrin-like protein. Molecular and Biochemical Parasitology 135, 31–38. http://dx.doi.org/10.1111/j.1462-5822.2005.00487.x.CrossRefGoogle ScholarPubMed
Marion, S., Laurent, C. and Guillén, N. (2005). Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach. Cellular Microbiology 7, 1504–1518. http://dx.doi.org/10.1111/j.1462-5822.2005.00573.x.CrossRefGoogle ScholarPubMed
Mendoza, L., Orozco, E., Rodríguez, M. A., García-Rivera, G., Sánchez, T., García, E. and Gariglio, P. (2003). Ehp53, an Entamoeba histolytica protein, antecesor of the mammalian tumor suppressor p53. Microbiology 149, 885–893. http://dx.doi.org/10.1099/mic.0.25892-0.CrossRefGoogle Scholar
Mosammaparast, N. and Pemberton, L. F. (2004). Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends in Cell Biology 14, 547–556. http://dx.doi.org/10.1016/j.tcb.2004.09.004.CrossRefGoogle ScholarPubMed
O'Reilly, A. J., Dacks, J. B. and Field, M. C. (2011). Evolution of the karyopherin-β family of nucleocytoplasmic transport factors; ancient origins and continued specialization. PLoS One 6, e19308. http://dx.plos.org/10.1371/journal.pone.0019308.CrossRefGoogle ScholarPubMed
Pritt, B. S. and Clark, C. G. (2008). Amebiasis. Mayo Clinic Proceedings 83, 1154–1160. http://dx.doi.org/10.4065/83.10.1154.CrossRefGoogle ScholarPubMed
Rodríguez, E., Aburjania, N., Priedigkeit, N. M., DiFeo, A. and Martignetti, J. A. (2010). Nucleo-cytoplasmic localization domains regulate Krüppel-like factor 6 (KLF6) protein stability and tumor supresor function. PLoS One 5, e12639. http://dx.plos.org/10.1371/journal.pone.0012639.CrossRefGoogle Scholar
Saarikangas, J., Zhao, H. and Lappalainen, P. (2010). Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides. Physiological Reviews 90, 259–289. http://dx.doi.org/10.1152/physrev.00036.2009.CrossRefGoogle ScholarPubMed
Sahoo, N., Labruyère, E., Bhattacharya, S., Sen, P., Guillén, N. and Bhattacharya, A. (2004). Calcium binding protein 1 of the protozoan parasite Entamoeba histolytica interacts with actin and is involved in cytoskeleton dynamics. Journal of Cell Science 117, 3625–3634. http://dx.doi.org/10.1242/jcs.01198.CrossRefGoogle ScholarPubMed
Shen, V., Liu, H., Liu, S. W., Jiao, X. and Kiledjan, M. (2008). DcpS scavenger decapping enzyme can modulate pre-mRNA splicing. RNA 14, 1132–1142. http://dx.doi.org/10.1261/rna.1008208.CrossRefGoogle ScholarPubMed
Singh, D., Naik, S. and Naik, S. (2004). Role of cysteine proteinase of Entamoeba histolytica in target cell death. Parasitology 129, 127–135. http://dx.doi.org/10.1017/S0031182004005451.CrossRefGoogle ScholarPubMed
Stanley, S. L. Jr. (2003). Amoebiasis. Lancet 361, 1025–1034. http://dx.doi.org/10.1016/S0140-6736(03)12830-9.CrossRefGoogle ScholarPubMed
Taagepera, S., McDonald, D., Loeb, J. E., Whitaker, L. L., McElroy, A. K., Wang, J. Y. and Hope, T. J. (1998). Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase. Proceedings of the National Academy of Sciences, USA 95, 7457–7462.CrossRefGoogle ScholarPubMed
Tovy, A., Siman, Tov. R., Gaentzsch, R., Helm, M. and Ankri, S. (2010). A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2) activity. PloS Pathogens 6, e1000775. http://dx.plos.org/10.1371/journal.ppat.1000775.CrossRefGoogle ScholarPubMed
Uribe, R. and Jay, D. (2009). A review of actin binding proteins: new perspectives. Molecular Biology Reports 36, 121–125. http://dx.doi.org/10.1007/s11033-007-9159-2.CrossRefGoogle ScholarPubMed
Van Etten, R. A., Jackson, P. and Baltimore, D. (1989). The mouse type IV c-abl gene product is a nuclear protein, and activation of transforming ability is associated with cytoplasmic localization. Cell 58, 669–678. http://dx.doi.org/10.1016/0092-8674(89)90102-5.CrossRefGoogle ScholarPubMed
Vargas, M., Sansonetti, P. and Guillén, N. (1996). Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica. Molecular Microbiology 22, 849–857. http://dx.doi.org/10.1046/j.1365-2958.1996.01535.x.CrossRefGoogle ScholarPubMed
Vázquez, J., Franco, E., Reyes, G. and Meza, I. (1995). Characterization of adhesion plates induced by the interaction of Entamoeba histolytica trophozoites with fibronectin. Cell Motility and the Cytoskeleton 32, 37–45. http://dx.doi.org/10.1002/cm.970320105.CrossRefGoogle ScholarPubMed
Virel, A. and Backman, L. (2004). Molecular evolution and structure of alpha-actinin. Molecular biology and evolution 21, 1024–1031. http://dx.doi.org/10.1093/molbev/msh094.CrossRefGoogle ScholarPubMed
Virel, A. and Backman, L. (2006). Characterization of Entamoeba histolytica alpha-actinin. Molecular and Biochemical Parasitology 145, 11–17. http://dx.doi.org/10.1016/j.molbiopara.2005.09.003.CrossRefGoogle ScholarPubMed
Washington, R. W. and Knecht, D. A. (2008). Actin binding domains direct actin-binding proteins to different cytoskeletal locations. BMC Cell Biology. 9, 10. doi:10.1186/1471-2121-9-10.CrossRefGoogle ScholarPubMed
WHO/PAHO/UNESCO. (1997). A Consultation with Experts on Amoebiasis. Epidemiological Bulletin No. 18, Mexico City, Mexico.Google Scholar
Wu, X., Yoo, Y., Okuhama, N. N., Tucker, P. W., Liu, G. and Guan, J. L. (2006). Regulation of RNA-polymerase-II-dependent transcription by N-WASP and its nuclear-binding partners. Nature Cell Biology 8, 756–763. http://dx.doi.org/10.1038/ncb1433.CrossRefGoogle ScholarPubMed
Xu, D., Farmer, A. and Chook, Y. M. (2010). Recognition of nuclear targeting signals by Karyopherin-β proteins. Current Opinion in Structural Biology 20 782–790. http://dx.doi.org/10.1016/j.sbi.2010.09.008.CrossRefGoogle ScholarPubMed
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