摘要
生物大分子的核质转运是由受体importinβ蛋白家族成员介导完成的。importin 13(imp13,或者lg12、IPO13)是importinβ蛋白家族的一个成员,它的表达和功能是受发育调节。Northern Blot和免疫组化实验均表明IPO13在脑的表达水平比其他组织都高。imp13是目前所知的哺乳动物细胞中唯一具有双向转运功能的受体蛋白。
为了进一步研究imp13的功能,在本研究中,我们用羧基末端imp 13(aminoacid 307-963)(C-imp13)作为诱饵蛋白,用酵母双杂交系统筛选人胚胎脑cDNA文库中能够与其相互作用的蛋白,发现Arx蛋白质能与羧基末端imp 13相互作用。为了确定这个相互作用,我们构建了能表达带有GST和His_6标签的全长Arx的原核表达质粒。通过GST pull-down和免疫共沉淀实验,我们发现,像C端Arx与imp13相互作用一样,全长Arx也能与imp13(外源和内源)相互作用。由于imp13能够与Arx相互作用,我们推测Arx有可能是imp13的入核底物。
Arx(Aristaless-related homeobox)蛋白是成对同源结构域蛋白家族的一个成员。ARX基因的突变和多种X—连锁的疾病有关。研究发现Arx对前脑,胰腺和睾丸的发育起重要作用。为了进一步研究Arx蛋白的功能,我们构建了表达Arx-GST融合蛋白质的表达载体pGEX4T2-F-Arx。F-Arx蛋白纯化后做抗原,免疫兔子,血清经纯化和验证后,获得了Arx多克隆抗体。
Nulceocytoplasmic transport of macromolecules is mediated by soluble transport receptors,namely importinβsuperfamily proteins.Importin 13(Imp13,also known as lg12、IPO13),is a member of the importinβsuperfamily,Its expression and function are developmentally regulated.Both Northern Blot analysis and microarray analysis show that the expression level is higher in the brain than in other tissues.
To study the function of imp13,we attempted to identify its interacting proteins from a human fetal brain cDNA library by Yeast Two-Hybrid screening using C-imp13(amino acid 307-963) as the bait.We found that Arx protein can interact with C-imp13.To confirm this interaction,we expressed full-length of mouse Arx tagged with either His_6 or GST in bacteria.Like the interaction of the C-terminal Arx fragment with imp13,full-length Arx binds imp 13 as judged by GST-pull down assays.Equivalent observations were made by co-immunoprecipitation assays.Since both endogenous imp13 and overexpressed imp13 bind Arx,we suggest that Arx is an imp13 import cargo.
The Arx protein(Aristaless-related homeobox) is a member of the paired class of homeoproteins.ARX gene mutations are related with many X-chromosome linked disorders.Many research has indicated that it is crucial for its functions in forebrain, pancreases and testes development.To study the function of Arx protein,we construct a vector called pGEX4T2-F-Arx which can express the fused protein GST-Arx.By immunizing rabbits with the purified recombinant protein,antiserum was obtain.After purification and verification,we gain the polyclonal antibody of Arx.
引文
[1] Weis K. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell [J], 2003, 112:441
[2] Lucy F. Pemberton , Bryce M. Paschal..Mechanism of Receptor-Mediated Nucleaar Import and Nuclear Export [J]. Traffic, 2005, 6:187-198
[3] Soo Jae Lee, Toshihiro Sekimoto, Eiki Yamashita, Emi Nagoshi, Atsushi Nakagawa, Naoko Imamoto, Masato Yoshimura, Hiroaki Sakai, Khoon Tee Chong, Tomitake Tsukihara, Yoshihiro Yoneda. The Structure of Importin-β Bound to SREBP-2: Nuclear Import of a Transcription Factor [J]. Science 302 (5650):1571-1575
[4] Gino Cingolani, Carlo Petosa, Karsten Weis and Christoph W. Muller.Structure of importin- β bound to the IBB domain of importin-α[J]. Nature 399:221 -229.
[5] Chook YM, Bolbel G. Structure of the nuclear transport complex karyopherin- beta2 -Ran x GooNHp [J]. Nature, 1999, 399(6733):230-237.
[6] Bayliss R, Litlewood T, Stewart M. Structural basis ofr the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking[J]. Cell, 2000, 102(1):99-108.
[7] Zhang C, Sweezey NB, Gagnon S, Muskat B, Koehler D, Post M, Kaplan F. A novel karyopherinbhomolog is developmentally and hormonally regulated in fetal lung [J]. Am J Respir Cell Mol Biol, 2000, 22(4):451 -459
[8] Ploski JE, Shamsher MK. Radu A. Paired-typed homeodomain transcription factors are imported into the nucleus by karyopherin13 [J]. Mol Cell Biol, 2004, 24(11):4824- 4834
[9] Mingot JM, Kostka S, Kraft T, Hartmann E, Gorlich D.Importin13: a novel mediator of nuclear import and export[J].EMBO J.2001, 20( 14):3685-3694.
[10] Kahle J, Baake M, Doenecke D, Albig W. Subunits of the heterotrimeric transcription factor NF-Y are imported into the nucleus by distinct pathways involving importin beta and importin13[J]. Mol Cell Biol, 2005, 25(13):5339-5354
[11] Tao T, Lan J, Lukacs GL, Hache R, Kaplan F. Importin 13 regulates nuclear import of the glucocoricoid receptor in airway ephithelial cells [J]. Am J Respir Cell Mol Biol, 2006, 35(6):668-680
[12] Freedman N, Yamamoto K. Importin7 and importin α /importin β are nuclear import receptors for the glucocorticoid receptor[J]. Mol Biol Cell, 2004, 15:2276-2286.
[13] Savory JG, Hsu B, Laquian IR, Giffin W, Reich T, Hache RJ.Lefebvre YA. Discrimination between NL-1 and NL2-mediated nuclear localization of the glucocorticoid receptor [J]. Mol Cell Biol, 1999, 19:1025-1037.
[14] Mingot JM, Kostka S, Kraft T, Hartmann E, Gorlich D.Importin13: a novel mediator of nuclear import and export[J] .EMBO J.2001, 20( 14):3685-3694.
[15] Yamaguchi Y L, Tanaka SS. Stage-specific Importin 13 activity influences meiosis of germ cells in the mouse[J]. Developmental Biology, 2006, (11):5-8.
[16] Ploski JE, Shamsher MK, Radu A. Paired-typed homeodomain transcription factors are imported into the nucleus by karyopherin13[J]. Mol Cell Biol, 2004, 24(11):4824- 4834.
[17] Kahle J, Baake M, Doenecke D, Albig W. Subunits of the heterotrimeric transcription factor NF-Y are imported into the nucleus by distinct pathways involving importin beta and importin 13[J]. Mol Cell Biol, 2005, 25(13):5339-5354.
[18] Tao T, Lan J, Lukacs GL, Hache R, Kaplan F. Importin 13 regulates nuclear import of the glucocoricoid receptor in airway ephithelial cells [J]. Am J Respir Cell Mol Biol, 2006, 35(6):668-680
[19] Liang, J., Ke, G., You, W., Peng, Z., Jie Lan, Markus Kalesse , Alan M. Tartakoff, Feige Kaplan , Tao Tao. Interaction between importin 13 and myopodin suggests a nuclear import pathway for myopodin[J]. Mol Cell Biochem (2008) 307:93-100
[20]Blair, H.J., Reed, V., Gormally, E., Wilson, J.B., Novak, J., McInnes, R.R., Phillips, S.J., Taylor, B.A., and Boyd, Y. 2002.Positioning of five genes (CASK, ARX, SAT, IMAGE cDNAs 248928 and 253949) from the human X chromosome short arm with respect to evolutionary breakpoints on the mouse X chromosome[J]. Mamm. Genome 11:710-712.
[21] Miura, H., Yanazawa, M., Kato, K., and Kitamura, K. (1997) Expression of a novel aristaless related homeobox gene 'ARX' in the vertebrate telencephalon, diencephalon and floor plate[J]. Mech. Dev., 65, 99-109.
[22]Stromme P, Mangelsdorf ME, Shaw MA et al: Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy[J]. Nat Genet 2002; 30:441- 445.
[23]Bienvenu T, Poirier K, Friocourt G et al: ARX, a novel Prd-classhomeobox gene highly expressed in the telencephalon, is mutated in X-linked mental retardation [J]. Hum Mol Genet 2002; 11:981-991.
[24] Meijlink, F., Beverdam, A., Brouwer, A., Oosterveen, T.C. & Berge, D.T.(I999) Vertebrate aristaless-related genes[J]. Int J. Dev. Biol., 43, 651 - 663.
[25] Ohira R, Zhang YH, Guo W, Dipple K, Shih SL, Doerr J, Huang BL, Fu LJ, Abu-Khalil A, Geschwind D, McCabe ER: Human ARX gene: genomic characterization and expression[J].Mol Genet Metab 2002, 77:179-188.
[26]Bejerano G, Pheasant M, Makunin I, Stephen S, Kent WJ.Mattick JS, Haussler D: Ultraconserved elements in the human genome[J]. Science 2004, 304:1321-1325.
[27]Collombat P, Hecksher-Sorensen J, Broccoli V, Krull J, Ponte I.Mundiger T, Smith J, Gruss P, Serup P, Mansouri A: The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the a- and b-cell lineages in the mouse endocrine pancreas[J].Development 2005, 132:2969-2980.
[28]Collombat P, Mansouri A, Hecksher-Sorensen J, Serup P, Krull J.Gradwohl G, Gruss P: Opposing actions of Arx and Pax4 in endocrine pancreas development[J]. Genes Dev 2003, 17:2591-2603.
[29] Elena Colombo, Rossella Galli, Giulio Cossu, Jozef Gecz and Vania Broccol: Mouse Orthologue of ARX, a Gene Mutated in Several X-Linked Forms of Mental Retardation and Epilepsy, Is a Marker of Adult Neural Stem Cells and Forebrain GABAergic Neurons[J]. Development dynamics 2004, 231:631 - 639.
[30]Lavdas, A.A., Grigoriou, M., Pachnis, V. & Pamavelas, J.G. (1999) The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex[J]. Neurosci., 19, 7881 - 7888.
[31] Poirier, K., Van Esch, H., Friocourt, G., Saillour, Y., Bahi, N., Backer, S., Souil.E., Castelnau-Ptakhine, L., Beldjord, C., Francis, F., Bienvenu, T. & Chelly, [J]. (2004) Neuroanatomical distribution of ARX in brain and its localization in GABAergic neurons. Brain Res. Mol. Brain Res., 122, 35 - 46.
[32]Marin, O. & Rubenstein, J.L. (2003) Cell migration in the forebrain[J]. Annu. Rev.Neuro sci, 26, 441 - 483.
[33] Szostak, J.W. In vitro genetics. Trends Biochem[J]. Sci. 1992; 17(3):89-93.
[34] Banerjee-Basu S, Baxevanis AD: Molecular evolution of the homeodomain family of transcription factors[J]. Nucleic Acids Res 2001, 29:3258-3269.
[35] Seufert DW, Prescott NL, El-Hodiri HM: Xenopus aristalessrelated homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain development[J]. Dev Dyn 2005, 232:313-324.
[36] Simeone, A., D' Apice, M.R., Nigro, V., Casanova, J., Graziani, F., Acampora, D. Avantaggiato, V. (1994) Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and Drosophila[J].Neuron, 13, 83 - 101.
[37] Hemmati-Brivanlou, A., de la Torre, J.R., Holt, C.and Harland R.M. (1991)Cephalic expression and molecular characterization of Xenopus En-2[J].Development, 111, 715-724.
[38]McKenzie O, Ponte I, Mangelsdorf M, Finnis M, Stifani S, Ge' cz J.Broccoli V: Mutations in the aristaless-related homeobox gene ARX affect its transcription repression activity[J]. J Biol Chem 2006. in press.
[39]. Kitamura, K., Yanazawa, M., Sugiyama, N., Miura, H., Iizuka-Kogo, A., Kusaka, M., Omichi, K., Suzuki, R., Kato-Fukui, Y., Kamiirisa, K., Matsuo.M., Kamijo, S.I., Kasahara, M., Yoshioka, H., Ogata, T., Fukuda, T., Kondo.I., Kato, M., Dobyns, W.B., Yokoyama, M. & Morohashi, K.I. (2002)Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans[J]. Nat.Genet., 32, 359 - 369.
[40]Seufert, D.W., Prescott, N.L. & El-Hodiri, H.M. (2005) Xenopus aristalessrelated homeobox (xArx) gene product functions as both a transcriptional activator and repressor in forebrain development[J]. Dev. Dyn., 232, 313-324.
[41]E1-Horidi, H.M., Qi, X.L. & Seufert, D.W. (2003) The Xenopus arx gene is expressed in the developing rostral forebrain. Dev. Genes Evol., 212, 608 - 612.
[42] Muenke, M. & Beachy, P. (2000) Genetics of ventral forebrain development and holoprosencephaly[J]. Curr. Opin. Genet. Dev., 10, 262 - 269.
[43]Anderson, S.A., Eisenstat, D.D., Shi, L. & Rubenstein, J.L.R. (1997)Intemeuron migration from basal forebrain to neocortex: dependence on Dlx genes[J]. Science, 278, 47 - 476.
[44]Lavdas, A.A., Grigoriou, M., Pachnis, V. & Parnavelas, J.G. (1999) The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex. [J] Neurosci., 19, 7881 - 7888.
[45]Wichterle, H., Tumbull, D.H., Nery, S., Fishell, G. & Alvarez-Buylla, A. (2001)In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain[J]. Development, 128, 3759-3771.
[46]Sussel, L., Marin, O., Kimura, S. & Rubenstein, J.L.R. (1999) Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum[J]. Development, 126, 3359 - 3370.
[47]. Anderson, S.A., Marin, O., Horn, C, Jennings, K. & Rubenstein, J.L. (2001 )Distinct cortical migrations from the medial and lateral ganglionic eminences[J]. Development, 128, 353 - 363.
[48] Cobos I, Broccoli V, John L.R. et al. The Vertebrate Ortholog of Aristaless Is Regulated by Dlx Genes in the Developing Forebrain[J].(2005) The Journal Of Comparative Neurology 483:292 - 303.
[49] Friocourt G, Poirier K, Rakic S, John G et al: The role of ARX in cortical development[J].2006, 23: 869 - 876.
[50] Chenn, A. & McConnell, S.K. (1995) Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis[J]. Cell, 82, 631-641.
[51]Takahashi, T., Nowakowski, R.S. & Caviness, V.S.J. (19%) The leaving or Q fraction of the murine proliferative epithelium: a general model of neocortical neurogenesis[J]. Neurosci., 16, 6183 - 6196.
[52]Miyata, T., Kawaguchi, A., Okano, H. & Ogawa, M. (2001) Asymmetric inheritance of radial glial fibers by cortical neurons[J]. Neuron, 31, 727 - 741.
[53]. Noctor, S.C., Flint, A.C., Weissman, T.A., Dammerman, R.S. & Kriegstein, A.R. (2001) Neurons derived from radial glial cells establish radial units in neocortex. [J] Nature, 409, 714 - 720.
[54]Sanada, K. & Tsai, L.H. (2005) G protein bc subunits and AGS3 control spindle orientation and asymmetric cell fate of cerebral cortical progenitors[J].Cell, 122, 119 - 131.
[55]Betschinger, J. & Knoblich, J.A. (2004) Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates[J]. Curr. Biol., 14, R674 - R685.
[56] Melkman, T. & Sengupta, P. (2005) Regulation of chemosensory and GABAergic motor neuron development by the C. elegans Aristaless/Arx homolog alrl[J]. Development, 132, 1935 - 1949
[57]Sarafi-Reinach, T.R., Melkman, T., Hobert, O. & Sengupta, P. (2001) The lin-11 LIM homeobox gene specifies olfactory and chemosensory neuron fates in C.elegans[J]. Development, 128, 3269 - 3281.
[58]Pueyo, J.I. & Couso, J.P. (2004) Chip-mediated partnerships of the homeodomain proteins Bar and Aristaless with the LIM-HOM proteins Apterous and Lim1 regulate distal leg development[J]. Development, 131, 3107 - 3120.
[59]Millonig, J.H., Millen, K.J. & Hatten, M.E. (2000) The mouse Dreher gene Lmx1a controls formation of the roof plate in the vertebrate CNS. [J] Nature, 403, 764 - 769.
[60]Stuhmer, T., Puelles, L., Ekker, M. & Rubenstein, J.L. (2002) Expression from a Dlx gene enhancer marks adult mouse cortical GABAergic neurons[J]. Cereb.Cortex, 12, 75 - 85.
[61] Adrian, T.E., Bloom, S.R., Hermansen, K., and Iversen, J. 1978. Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas[J]. Diabetologia 14:413 - 417.
[62]Roncoroni, L., Violi, V., Montanari, M., and Muri, M. 1983.Effect of somatostatin on exocrine pancreas evaluated on a total external pancreatic fistula of neoplastic origin[J]. Am. J.Gastroenterol. 78:425 - 428.
[63]Csaba, Z. and Doumaud, P. 2001. Cellular biology of somatostatin receptors[J], Neuropeptides 35: 1 - 23. [64]Slack JM (1995) Developmental biology of the pancreas[J]. Development 121: 1569-1580
[65] Wilson ME , Scheel D , German MS (2003) Gene expression cascades in pancreatic development[J]. Mech Develop 120:65-80
[66] Jensen J (2004) Gene regulatory factors in pancreatic development[J]. Develop Dynam 229: 176-200
[67]Gu G, Dubauskaite J, Melton DA (2002) Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors[J]. Development 129: 2447-2457
[68]Jensen J (2004) Gene regulatory factors in pancreatic development[J]. Develop Dynam 229: 176-200
[69]Ahlgren, U., Jonsson, J., Jonsson, L., Simu, K. and Edlund, H. (1998). β-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the β-cell phenotype and maturity onset diabetes[J]. Genes Dev. 12, 1763-1768.
[70]Mansouri, A., St-Onge, L. and Grass, P. (1999). Role of Genes in Endodermderived Organs[J]. Trends Endocrinol. Metab. 10, 164-167.
[71]Sander, M., Sussel, L., Conners, J., Scheel, D., Kalamaras, J., Dela Cruz, F., Schwitzgebel, V., Hayes-Jordan, A. and German, M. (2000).Homeobox gene Nkx6. 1 lies downstream of Nkx2. 2 in the major pathway of β-cell formation in the pancreas. [J] Development 127, 5533-5540.
[72] Smith, S. B., Ee, H. C, Conners, J. R. and German, M. S. (1999). Paired homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development[J]. Mol. Cell Biol. 19, 8272-8280.
[73]Sosa-Pineda, B., Chowdhury, K., Torres, M., Oliver, G. and Grass, P.(1997). The Pax4 gene is essential for differentiation of insulin-producing β-cells in the mammalian pancreas[J]. Nature 386, 399-402.
[74]Sussel, L., Kalamaras, J., Hartigan-O'Connor, D. J., Meneses, J. J., Pedersen, R. A., Rubenstein, J. L. and German, M. S. (1998). Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic β-cells[J]. Development 125, 2213-2221.
[75] Noctor, S.C., Flint, A.C., Weissman, T.A., Dammerman, R.S. & Kriegstein, A. R. (2001) Neurons derived from radial glial cells establish radial units in neocortex. [J] Nature, 409, 714-720
[76] Yu Quan, Zhi-Liang Ji, Xiao Wang, Alan M. Tartakoff1 and Tao Tao.(2008) Evolutionary and transcriptional Analysis of Karyopherin β Superfamily Proteins. MCP Papers in Press.
