斑马鱼分拣蛋白SNX10基因功能研究
摘要
SNXs(sorting nexins)是近年开始研究的一个基因家族,以包含PX(Phoxhomology)结构域、定位于早期内涵体、参与细胞内蛋白分拣为特征。从真菌酵母到人类广泛表达,己知部分SNXs参与EGFR(epidermal growth factor receptor)内吞分拣,大多数家族成员功能尚未报道。文献报道人和鼠类SNX10在多种细胞系中诱导成泡,个体水平的生理功能未有研究。本文以斑马鱼为模型,研究斑马鱼dSNX10a和dSNX10b在斑马鱼早期胚胎发育过程中的生理功能,利用细胞转染、原位杂交、基因敲低等技术,证实斑马鱼dSNX10a是人类hSNX10的同源蛋白,参与斑马鱼早期左右不对称发育;dSNX10b是鱼类特有基因,参与心脏发育。斑马鱼dSNX10a和dSNX10b的氨基酸序列有61%的同源性,都定位于早期内涵体,却有着截然不同的细胞和生理水平的生物功能。研究结果将为进一步探讨两者分子结构与生物功能的关系奠定基础,也为研究SNXs家族基因功能提供了一种新的方法。
SNXs(sorting nexins) is a new gene family, featured with PX domain, localizing in early endosome, and playing a role in proteins sorting in eukaryotic cells. As a conservative gene family, they express from yeast to human. It is reported that some of them participate in EGFR (epidermal growth factor receptor) sorting, while functions of most of these proteins haven't been explored.
SNX10 of either human or mice induces large vacuoles in eukaryotic cell lines as reported, however its function in vivo is still a question. With the model organism zebrafish (Danio rerio), this study tried to illustrate physiological function of dSNX10a and dSNX10b in early development stages. Through cell lines transfection, ISH (in situ hybridization), and knock-down technologies, we demonstrated dSNX10a is the ortholog of human SNX10 (hSNX10), participating in Danio L/R asymmetry foundation, and dSNX10b is a fish specific gene, playing a role in Danio heart development. Although the two genes share high identity (61%), also both of them localize in early endosome, they function throughly differently in cell lines and in vivo. Our results is the basis to study the relationship between molecular structure and their function, and it could also be an attempt as a method to further explore the function of other SNXs families.
SNXs(sorting nexins) is a new gene family, featured with PX domain, localizing in early endosome, and playing a role in proteins sorting in eukaryotic cells. As a conservative gene family, they express from yeast to human. It is reported that some of them participate in EGFR (epidermal growth factor receptor) sorting, while functions of most of these proteins haven't been explored.
SNX10 of either human or mice induces large vacuoles in eukaryotic cell lines as reported, however its function in vivo is still a question. With the model organism zebrafish (Danio rerio), this study tried to illustrate physiological function of dSNX10a and dSNX10b in early development stages. Through cell lines transfection, ISH (in situ hybridization), and knock-down technologies, we demonstrated dSNX10a is the ortholog of human SNX10 (hSNX10), participating in Danio L/R asymmetry foundation, and dSNX10b is a fish specific gene, playing a role in Danio heart development. Although the two genes share high identity (61%), also both of them localize in early endosome, they function throughly differently in cell lines and in vivo. Our results is the basis to study the relationship between molecular structure and their function, and it could also be an attempt as a method to further explore the function of other SNXs families.
引文
[1] Qin, B., et al. Sorting nexins 10 induces giant vacuoles in mammalian cells. J Biol Chem, 2006, 281(48):36891-36896.
[2] Carlton, J., et al. Sorting nexins--unifying trends and new perspectives. Traffic, 2005, 6(2):75-82.
[3] Li-Fong Seet, Wan jin Hong. The Phox (PX) domain proteins and membrane traffic . Biochimica et Biophysica Acta, 2006(1761):878-896.
[4] Yue XU, Li-Fong SEET, Brendon HANSON and Wanjin HONG1. The Phox homology (PX) domain, a new player in phosphoinositide Signaling. Biochem. J, 2001(360):513-530.
[5] Kurten, R.C., D.L. Cadena, and G.N. Gill. Enhanced degradation of EGF receptors by a sorting nexins, SNX1. Science, 1996, 272(5264): 1008-1010.
[6] GμLlapalli, A., et al. A role for sorting nexins 2 in epidermal growth factor recepto r down-regulation: evidence for distinct functions of sorting nexins 1 and 2 in protein trafficking. Mol Biol Cell, 2004, 15(5):2143-2155.
[7] Traer. C.J., et al. SNX4 coordinates endosomal sorting of TfnR with dynein-media ted transport into the endocytic recycling compartment. Nat Cell Biol., 2007(9):370-380.
[8] Merino-Trigo, A., et al. Sorting nexins 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation. J Cell Sci, 2004, 117(26):6413-6424.
[9] Parks, W.T., et al. Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases. J Biol Chem, 2001, 276(22):19332-19339.
[10] Badour, K., et al. Interaction of the Wiskott-Aldrich syndrome protein with sorting nexins 9 is required for CD28 endocytosis and cosignaling in T cells. Proc Natl Acad Sci USA, 2007, 104(5): 1593-1598.
[11] Zheng, B., et al. Essential role of RGS-PX1 /sorting nexins 13 in mouse d evelopment and regulation of endocytosis dynamics. Proc Natl Acad Sci USA, 2006, 103(45):16776-16781.
[12] Choi, J.H., et al. Sorting nexins 16 regulates EGF receptor trafficking by phosphatidylinositol-3-phosphate interaction with the Phox domain. J Cell Sci, 2004, 117(18):4209-4218.
[13] van Kerkhof, P., et al. Sorting nexins 17 facilitates LRP recycling in the early endosome. Embo J., 2005, 24(16):2851-2861.
[14] Williams, R., et al. Sorting nexins 17 accelerates internalization yet retards degradation of P-selectin. Mol Biol Cell, 2004, 15(7):3095-3105.
[15] Schaff, U.Y., et al. SLIC-1/sorting nexins 20: A novel sorting nexins that directs subcellular distribution of PSGL-1. Eur J Immunol, 2008, 38(2):550-564.
[16] Lunn, M.L., et al. A unique sorting nexins regulates trafficking of potassium channels via a PDZ domain interaction. Nat Neurosci, 2007, 10(10): 1249-1259.
[17] Carolyn A. Worby and Jack E.Dixon. Sorting Out the Cellular Functions of Sorting nexins. Nat Reviews, 2002, 12(3):919-931.
[18] Schwarz, D.G., et al. Genetic analysis of sorting nexins 1 and 2 reveals aredundant and essential function in mice. Mol Biol Cell, 2002, 13(10): 3588-600.
[19] James Summerton. Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochimica et Biophysica Acta, 1999(1489):141- 158.
[20] JAMES SUMMERTON and DWIGHT WELLER. Morpholino Antisense Oligomers: Design, Preparation, and Properties.ANTISENSE & NUCLEIC ACID DRMG DEVELOPMENT, 1997(7): 187-195.
[21] Christine Thisse & Bernard Thisse. High-resolution in situ hybridization to whole-mount zebrafish embryos. NAT PROTOCOLS, 2008, 3(1):59-69.
[22] George Streisinger. A guide for the laboratory use of zebrafish Danio (Brachydanio) rerio. 2000, 5th Edition:3-12.
[23] Nasevicius, A. and S.C. Ekker. Effective targeted gene 'knockdown' in zeb rafish. Nat Genet, 2000, 26(2):216-230.
[24] Nobutaka Hirokawa, Yosuke Tanaka, et al. Nodal Flow and the Generation of Left-Right Asymmetry. Cell, 2006(125):33-45.
[25] Gilbert, S.F. Developmental Biology, 2003, 7th Edition: 156-157.
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[27] Harvey, R.P. Links in the left/right axial pathway. Cell, 1998(94):273-276.
[28] Yost, H.J. Diverse initiation in a conserved left-right pathway? Curr. Opin. Genet. Dev, 1999(9):422-426.
[29] Levin, M. Left-right asymmetry in embryonic development: a Compreh ensive review. Mech. Dev, 2005(122):3-25.
[30] Hamada, H. Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet, 2002(3): 103-113.
[31] Capdevila, J., Vogan, K.J., Tabin,C.J., et al. Mechanisms of left-right deter mination in vertebrates. Cell, 2000(101):9-21.
[32] Beddington, R.S.R, and Robertson, E.J. Axis development and early asymmetry of mammals. Cell, 1999(96):195—209.
[33] Buceta, J., Ibas, M., et al. Nodal cilia dynamics and the speci fication of the left/right axis in early vertebrate embryo development. Biophys. J., 2005(89):2199-2209.
[34] Essner, J.J., Vogan, K.J., et al. Conserved function for embryonic nodal cilia.Nature, 2002(418):37-38.
[35] Essner, J.J., Amack, J.D.,et al. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development, 2005(132): 1247-1260.
[36] Okada, Y, Takeda, S., et al. Mechanism of nodal flow: A conserved symmetry breaking event in left-right axis determination. Cell, 2005(121):633-644.
[37] Sampath, K., Cheng, A.M., et al. Functional differences among Xenopus nodal-related genes in left-right axis determination. Development, 1997(124):3293-3302.
[38] GN Serbedzija, JN Chen and MC Fishman. Regulation in the heart field of zebrafish. Development, 1998(6): 1095-1101.
[39] Olson EN, Srivastava D. Molecular pathways controlling heart development. Science, 1996(5262):671-676.
[40] Mark Peifer, PaμL Polakis. Wnt Signaling in Oncogenesis and Embryogenesis--a Look Outside the Nucleus. Science, 2000(287): 1606-1609.
[41] Andreas Wodarz. MECHANISMS OF WNT SIGNALING IN DEVELOPM ENT. Annual Review of Cell and Developmental Biology, 1998(14):59-88.
[42] M Tanaka, Z Chen, S. Bartunkova, et al. The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development, 1999(126):1269-1280.
[43] CM Jones, KM Lyons and BL Hogan. Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development, 1991(111):531-542.
[44] Q .Ying, J. Nichols, et al. BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3. Cell, 2003(115):281-292.
[45] Ornitz DM, Xu J, Colvin JS,et al. Receptor specificity of the fibroblast growth factor family. J Biol Chem., 1996, 271(25): 15292-15297.
[46] Szebenyi G, Fallon JF. Fibroblast growth factors as multifunctional signaling factors. Int Rev Cytol., 1999(185):45-106.
[47] Timothy A. McKinsey, Chun Li Zhang. MEF2: a calcium-dependent regulator of cell division, differentiation and death. Trends in Biochemical Sciences,2002(27):40-47.
[48] L J Ko and J D Engel .DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol., 1993(13):4011-4022.
[49] MICHAEL PARTRIDGE, ALEXANDRA VINCENT, et al. A Simple Method for Delivering Morpholino Antisense Oligos into the Cytoplasm of Cells. ANTISENSE & NUCLEIC ACID DRMG DEVELOPMENT, 1996(6): 169-175.
[2] Carlton, J., et al. Sorting nexins--unifying trends and new perspectives. Traffic, 2005, 6(2):75-82.
[3] Li-Fong Seet, Wan jin Hong. The Phox (PX) domain proteins and membrane traffic . Biochimica et Biophysica Acta, 2006(1761):878-896.
[4] Yue XU, Li-Fong SEET, Brendon HANSON and Wanjin HONG1. The Phox homology (PX) domain, a new player in phosphoinositide Signaling. Biochem. J, 2001(360):513-530.
[5] Kurten, R.C., D.L. Cadena, and G.N. Gill. Enhanced degradation of EGF receptors by a sorting nexins, SNX1. Science, 1996, 272(5264): 1008-1010.
[6] GμLlapalli, A., et al. A role for sorting nexins 2 in epidermal growth factor recepto r down-regulation: evidence for distinct functions of sorting nexins 1 and 2 in protein trafficking. Mol Biol Cell, 2004, 15(5):2143-2155.
[7] Traer. C.J., et al. SNX4 coordinates endosomal sorting of TfnR with dynein-media ted transport into the endocytic recycling compartment. Nat Cell Biol., 2007(9):370-380.
[8] Merino-Trigo, A., et al. Sorting nexins 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation. J Cell Sci, 2004, 117(26):6413-6424.
[9] Parks, W.T., et al. Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases. J Biol Chem, 2001, 276(22):19332-19339.
[10] Badour, K., et al. Interaction of the Wiskott-Aldrich syndrome protein with sorting nexins 9 is required for CD28 endocytosis and cosignaling in T cells. Proc Natl Acad Sci USA, 2007, 104(5): 1593-1598.
[11] Zheng, B., et al. Essential role of RGS-PX1 /sorting nexins 13 in mouse d evelopment and regulation of endocytosis dynamics. Proc Natl Acad Sci USA, 2006, 103(45):16776-16781.
[12] Choi, J.H., et al. Sorting nexins 16 regulates EGF receptor trafficking by phosphatidylinositol-3-phosphate interaction with the Phox domain. J Cell Sci, 2004, 117(18):4209-4218.
[13] van Kerkhof, P., et al. Sorting nexins 17 facilitates LRP recycling in the early endosome. Embo J., 2005, 24(16):2851-2861.
[14] Williams, R., et al. Sorting nexins 17 accelerates internalization yet retards degradation of P-selectin. Mol Biol Cell, 2004, 15(7):3095-3105.
[15] Schaff, U.Y., et al. SLIC-1/sorting nexins 20: A novel sorting nexins that directs subcellular distribution of PSGL-1. Eur J Immunol, 2008, 38(2):550-564.
[16] Lunn, M.L., et al. A unique sorting nexins regulates trafficking of potassium channels via a PDZ domain interaction. Nat Neurosci, 2007, 10(10): 1249-1259.
[17] Carolyn A. Worby and Jack E.Dixon. Sorting Out the Cellular Functions of Sorting nexins. Nat Reviews, 2002, 12(3):919-931.
[18] Schwarz, D.G., et al. Genetic analysis of sorting nexins 1 and 2 reveals aredundant and essential function in mice. Mol Biol Cell, 2002, 13(10): 3588-600.
[19] James Summerton. Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochimica et Biophysica Acta, 1999(1489):141- 158.
[20] JAMES SUMMERTON and DWIGHT WELLER. Morpholino Antisense Oligomers: Design, Preparation, and Properties.ANTISENSE & NUCLEIC ACID DRMG DEVELOPMENT, 1997(7): 187-195.
[21] Christine Thisse & Bernard Thisse. High-resolution in situ hybridization to whole-mount zebrafish embryos. NAT PROTOCOLS, 2008, 3(1):59-69.
[22] George Streisinger. A guide for the laboratory use of zebrafish Danio (Brachydanio) rerio. 2000, 5th Edition:3-12.
[23] Nasevicius, A. and S.C. Ekker. Effective targeted gene 'knockdown' in zeb rafish. Nat Genet, 2000, 26(2):216-230.
[24] Nobutaka Hirokawa, Yosuke Tanaka, et al. Nodal Flow and the Generation of Left-Right Asymmetry. Cell, 2006(125):33-45.
[25] Gilbert, S.F. Developmental Biology, 2003, 7th Edition: 156-157.
[26] Kaufman, M.H.The Atlas of Mouse Development. 1992, 1st edition:55-57.
[27] Harvey, R.P. Links in the left/right axial pathway. Cell, 1998(94):273-276.
[28] Yost, H.J. Diverse initiation in a conserved left-right pathway? Curr. Opin. Genet. Dev, 1999(9):422-426.
[29] Levin, M. Left-right asymmetry in embryonic development: a Compreh ensive review. Mech. Dev, 2005(122):3-25.
[30] Hamada, H. Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet, 2002(3): 103-113.
[31] Capdevila, J., Vogan, K.J., Tabin,C.J., et al. Mechanisms of left-right deter mination in vertebrates. Cell, 2000(101):9-21.
[32] Beddington, R.S.R, and Robertson, E.J. Axis development and early asymmetry of mammals. Cell, 1999(96):195—209.
[33] Buceta, J., Ibas, M., et al. Nodal cilia dynamics and the speci fication of the left/right axis in early vertebrate embryo development. Biophys. J., 2005(89):2199-2209.
[34] Essner, J.J., Vogan, K.J., et al. Conserved function for embryonic nodal cilia.Nature, 2002(418):37-38.
[35] Essner, J.J., Amack, J.D.,et al. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development, 2005(132): 1247-1260.
[36] Okada, Y, Takeda, S., et al. Mechanism of nodal flow: A conserved symmetry breaking event in left-right axis determination. Cell, 2005(121):633-644.
[37] Sampath, K., Cheng, A.M., et al. Functional differences among Xenopus nodal-related genes in left-right axis determination. Development, 1997(124):3293-3302.
[38] GN Serbedzija, JN Chen and MC Fishman. Regulation in the heart field of zebrafish. Development, 1998(6): 1095-1101.
[39] Olson EN, Srivastava D. Molecular pathways controlling heart development. Science, 1996(5262):671-676.
[40] Mark Peifer, PaμL Polakis. Wnt Signaling in Oncogenesis and Embryogenesis--a Look Outside the Nucleus. Science, 2000(287): 1606-1609.
[41] Andreas Wodarz. MECHANISMS OF WNT SIGNALING IN DEVELOPM ENT. Annual Review of Cell and Developmental Biology, 1998(14):59-88.
[42] M Tanaka, Z Chen, S. Bartunkova, et al. The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development, 1999(126):1269-1280.
[43] CM Jones, KM Lyons and BL Hogan. Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development, 1991(111):531-542.
[44] Q .Ying, J. Nichols, et al. BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3. Cell, 2003(115):281-292.
[45] Ornitz DM, Xu J, Colvin JS,et al. Receptor specificity of the fibroblast growth factor family. J Biol Chem., 1996, 271(25): 15292-15297.
[46] Szebenyi G, Fallon JF. Fibroblast growth factors as multifunctional signaling factors. Int Rev Cytol., 1999(185):45-106.
[47] Timothy A. McKinsey, Chun Li Zhang. MEF2: a calcium-dependent regulator of cell division, differentiation and death. Trends in Biochemical Sciences,2002(27):40-47.
[48] L J Ko and J D Engel .DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol., 1993(13):4011-4022.
[49] MICHAEL PARTRIDGE, ALEXANDRA VINCENT, et al. A Simple Method for Delivering Morpholino Antisense Oligos into the Cytoplasm of Cells. ANTISENSE & NUCLEIC ACID DRMG DEVELOPMENT, 1996(6): 169-175.