酵母双杂交系统筛选与Myosin X相互作用的蛋白质
|
摘要
酵母双杂交系统是目前研究蛋白质相互作用的重要手段之一,此方法具有高灵敏度、相对简单、快捷及不经蛋白纯化即可获得蛋白编码区的优点。近年来,酵母双杂交技术在研究蛋白质相互作用、筛选新蛋白、研究蛋白功能等方面发挥了重要作用。
肌球蛋白X(Myosin X, Myo X)是仅表达于脊椎动物中的非传统肌球蛋白,其尾部的MyTH4和FERM结构域可与多种功能蛋白相结合。这些蛋白与丝足的形成、神经元的突起生长与投射、内皮细胞的迁移、细胞的粘附及有丝分裂中纺锤体的形成等过程密切相关。因此Myo X在脊椎动物细胞中发挥了重要作用。
为了进一步深入的研究Myo X在脊椎动物体内的作用,本研究利用酵母双杂交技术,以Myo X的尾部结构域中的MyTH4和FERM结构为诱饵,对人胎脑cDNA文库进行了筛选。实验经Leu、Trp、His、Ade四种氨基酸营养缺陷型培养基筛选以及β-半乳糖苷酶活性分析,共获得112个阳性克隆,经过反复传代四种氨基酸营养缺陷型培养基筛选和β-半乳糖苷酶活性分析,去除一个酵母菌株中含有两个cDNA文库质粒的可能,再经过回交实验验证,最大程度的保证了酵母双杂交结果的准确性。从酵母菌株中提取质粒,转入大肠杆菌中,以氨苄青霉素(Amp)抗性筛选靶质粒,对部分阳性克隆的插入片段进行测序,在GENE BANK进行比对后分类,从分类结果可知与Myo X相互作用的蛋白涉及参与细胞迁移蛋白、细胞骨架蛋白、参与物质运输蛋白、参与基因表达调节的蛋白、参与信号转导蛋白等,为深入了解Myo X在生理上的新功能奠定基础。
Yeast two-hybrid system is one of the most important methods to study protein-protein interaction. The system is sensitive, simple and rapid, which the sequence of target proteins can be obtained directly. For the last few years, yeast two-hybrid system is used to study protein-protein interaction, screen the novel proteins and study the function of protein.
Myosin X is one of the members in unconventional myosin super-family and expresses at low concentrations in most vertebrate, which is relative to filopodial formation, neuronal growth, cell migration, cell adhesion and the spindle formation of mitosis. In this study, yeast two-hybrid technique was used to identify proteins interacting with Myo X. Here, the MyTH4 domain and FERM domain of Myo X tail are a bait to screen the human fetal brain cDNA library. Plating the cells on SD without histidine, tryptophan, leucine and adenine to assay the co-transformants to express the report genes and detect the expression of lac Z gene, a total of 112 positive clones were obtained, then, the positive clones were cultured in SD medium without histidine, tryptophan,leucine and adenine five times to remove the false clones. The backcross experiment was employed to ensure the results. The plasmid was extracted from yeast, and then it was transformed into E. coli and the target plasmid was obtained by selective culture. The cDNA segment of the positive clones were sequenced and compared with GENE BANK. The proteins screened in this study were probably involved in cell migration, cell adhesion, cystoskeleton rearrangement, protein transportation, regulation of gene expression and signal transduction.
肌球蛋白X(Myosin X, Myo X)是仅表达于脊椎动物中的非传统肌球蛋白,其尾部的MyTH4和FERM结构域可与多种功能蛋白相结合。这些蛋白与丝足的形成、神经元的突起生长与投射、内皮细胞的迁移、细胞的粘附及有丝分裂中纺锤体的形成等过程密切相关。因此Myo X在脊椎动物细胞中发挥了重要作用。
为了进一步深入的研究Myo X在脊椎动物体内的作用,本研究利用酵母双杂交技术,以Myo X的尾部结构域中的MyTH4和FERM结构为诱饵,对人胎脑cDNA文库进行了筛选。实验经Leu、Trp、His、Ade四种氨基酸营养缺陷型培养基筛选以及β-半乳糖苷酶活性分析,共获得112个阳性克隆,经过反复传代四种氨基酸营养缺陷型培养基筛选和β-半乳糖苷酶活性分析,去除一个酵母菌株中含有两个cDNA文库质粒的可能,再经过回交实验验证,最大程度的保证了酵母双杂交结果的准确性。从酵母菌株中提取质粒,转入大肠杆菌中,以氨苄青霉素(Amp)抗性筛选靶质粒,对部分阳性克隆的插入片段进行测序,在GENE BANK进行比对后分类,从分类结果可知与Myo X相互作用的蛋白涉及参与细胞迁移蛋白、细胞骨架蛋白、参与物质运输蛋白、参与基因表达调节的蛋白、参与信号转导蛋白等,为深入了解Myo X在生理上的新功能奠定基础。
Yeast two-hybrid system is one of the most important methods to study protein-protein interaction. The system is sensitive, simple and rapid, which the sequence of target proteins can be obtained directly. For the last few years, yeast two-hybrid system is used to study protein-protein interaction, screen the novel proteins and study the function of protein.
Myosin X is one of the members in unconventional myosin super-family and expresses at low concentrations in most vertebrate, which is relative to filopodial formation, neuronal growth, cell migration, cell adhesion and the spindle formation of mitosis. In this study, yeast two-hybrid technique was used to identify proteins interacting with Myo X. Here, the MyTH4 domain and FERM domain of Myo X tail are a bait to screen the human fetal brain cDNA library. Plating the cells on SD without histidine, tryptophan, leucine and adenine to assay the co-transformants to express the report genes and detect the expression of lac Z gene, a total of 112 positive clones were obtained, then, the positive clones were cultured in SD medium without histidine, tryptophan,leucine and adenine five times to remove the false clones. The backcross experiment was employed to ensure the results. The plasmid was extracted from yeast, and then it was transformed into E. coli and the target plasmid was obtained by selective culture. The cDNA segment of the positive clones were sequenced and compared with GENE BANK. The proteins screened in this study were probably involved in cell migration, cell adhesion, cystoskeleton rearrangement, protein transportation, regulation of gene expression and signal transduction.
引文
[1] Fields S and Song. A novel genetic system to detect protein–protein interactions [J]. Nature, 1989, 340 :245 - 246.
[2] Guthrie C & Fink, G R. Guide to yeast genetics and molecular biology[J]. In Methods in Enzymology (Academic Press, San Diego ), 1991, 194:1–932.
[3] Heslot, H & Gaillardin C, eds. Molecular Biology and Genetic Engineering of Yeasts[J]. CRC Press, 1992,18: 545-557.
[4] Chien, C.T. ,Bartel, P. L. , Sternglanz, R. & Fields, S. The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Nat. Acad. Sci, 1991, 88: 9578–9582.
[5] M Tanowitz and M von Zastrow. Identification of protein interactions by yeast two-hybrid screening and coimmunoprecipitation[J]. Methods MolBiol, 2004, 259 : 353.
[6] Garabet G. Toby, Erica A. Golemis. Using the Yeast Interaction Trapand Other Two-Hybrid Based Approaches to Study Protein-Protein Interactions [J]. Methods, 2001, 24 (3) : 201.
[7] Hope L. Lancero, Schryl Castaneda. Analysing protein–protein interactions of the Myxococcus xanthus Dif signalling pathway using the yeast two-hybrid system [J]. Microbiology, 2005, 151, 1535–154.
[8] Ilya G. Serebriiskii, Erica A. Golemis. Uses of lac Z to Study GeneFunction: Evaluation ofβ-Galactosidase Assays Employed in the Yeast Two-Hybrid System [J]. Analytical Biochemistry, 2000, 285(1): 1.
[9] Guarente, L.Strategies for the identification of interacting proteins. Proc Natl Acad Sci USA , 1993, 90:1639–1641.
[10] Estojak, J., Brent, R. & Golemis,E. A. Correlation of two-hybrid affnity data with in vitro measurements. Molecular and Cellular Biology, 1995,15: 5820-5829.
[11] Yang M, Wu Z & Fields S. Protein-peptide interactions analyzed with the yeast two-hybrid system. Nucleic Acid Res, 1995,23(7): 1152–1156.
[12] Wang Jun-Jie. Involvement of headless myosin X in the motility of immortalized gonadotropin-releasing hormone neuronal cells. Cell Biology International, 2009: 1-9.
[13] Sousa A D and R E Cheney, Myosin-X: a molecular motor at the cell's fingertips[J]. Trends Cell Biol, 2005, 15(10):533-5399.
[14] Homma k, and M Ikebe. Myosin X is a high duty ratio motor[J]. Biol Chem, 2005, 280: 29381-29391.
[15] Homma,k.,J.Saito,R.Ikebe,andM.Ikebe. Motor function and regulation of myosinX.J.Biol.Chem, 2001,276: 34348- 34354.
[16] Tokuo H and MIkebe, Myosin X transports Mena/VASP to the tip of filopodia [J]. Biochem Biophys Res Commun, 2004, 319(1): 214-220.
[17] Zhang H, Berg J S, Li Z, et al. Myosin-X provides a motor-based link between integrins and the cytoskeleton [J]. Nat Cell Biol, 2004, 6(6): 523-31.
[18] Chavrier P,May the force be with you:Myosin-X in phagocytosis[J]. Nat Cell Biol, 2002, 4(7): E169-71.
[19] Cox D, Berg J S, Cammer M, et al.,Myosin X is a downstream effector of PI(3)K during phagocytosis[J]. Nat Cell Biol, 2002, 4(7): 469-77.
[20] Pi X, Ren R, Kelley R,et al., Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors[J]. J Cell Biol, 2007, 179(7): 1569-82.
[21] Bohil A B, B W Robertson,and R E Cheney,Myosin-X is a molecular motor that functions in filopodiaformation[J].Proc Natl Acad Sci US A, 2006, 103(33): 12411-6.
[22] Bohil A B, B W Robertson,and R E Cheney,Myosin-X is a molecular motor that functions infilopodia for mation[J].Proc Natl Acad Sci USA, 2006, 103(33):12411-6.
[23] Tokuo H, K Mabuchi, and M Ikebe, The motor activity of myosin-X promotes actin fiber convergenceat the cell periphery to initiate filopodia formation[J]. J Cell Biol,2007, 179(2):229-38.
[24] Sarah Woolner, et al., Myosin-10 and actin filaments are essential for mitotic spindle function[J]. J Cell Biol, 2008:1-12.
[25] Toby GG, Gherraby W, Coleman TR, Golemis EA. A novel RING-finger protein, Human Enhancer of Invasion 10 (HEI10), alters mitotic progression through regulation of cyclin B levels[J]. Mol Cell Biol ,2003, 23:2109–2122.
[26] Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY, Kaiser BK, et al. The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases[J]. Trends in Cell Biol, 2000, 10:429–439.
[27] Mine N, Kurose K, Konishi H, et al. Fusion of a sequence from HEI10 (14q11) to the HMGIC gene at 12q15 in a uterine leiomyoma. Jpn[J]. Cancer Res, 2001,92: 135–139.
[28] Gronholm M, Muranen T, Toby GG, et al. A functional association between merlin and HEI10, a cell cycle regulator[J]. Oncogene, 2006.
[29] Evans DG, Sainio M, Baser ME. Neurofibromatosis type 2[J]. J Med Genet, 2000, 37: 897–904.
[30] Xiao GH, Gallagher R, Shetler J, et al. The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1expression[J]. Mol Cell Biol, 2005, 25: 2384–2394.
[31] Mahendra K. Singh, et al. HEI10 negatively regulates cell migration by inhibiting cyclin B/ Cdk1 and other promotility proteins[J]. Oncogene, 2007; 26(33): 4825–4832.
[32] Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments[J]. Cell, 2003, 112(4): 453-465.
[33] Plastino J, Sykes C. The actin slingshot, Curr Opin[J]. Cell Biol, 2005, 17(1): 62-66
[34] Huiwu Zhao, et al. UXT is a novel centrosomal protein essential for cell viability[J]. Molecular Biology of the Cell, 2005, 16: 5857–5865.
[35]Anderson S J, Wilkinson C J , Mayor T, et al. Proteomiccharacterization of the human centrosome by protein correlation profiling[J]. Nature, 2003,426,570–574.
[36] Schroer A, Schneider S, Ropers H & Nothwang HG. Cloning and characterization of UXT, a novel gene inhumanXp11,which is widely and abundantly expressed in tumortissue[J]. Genomics, 1999, 56, 340–343.
[37] R. McGilvray et al. UXT interacts with the transcriptional repressor protein EVI1 and suppresses cell transformation[J]. FEBS, 2007, 274 ,3960–3971.
[2] Guthrie C & Fink, G R. Guide to yeast genetics and molecular biology[J]. In Methods in Enzymology (Academic Press, San Diego ), 1991, 194:1–932.
[3] Heslot, H & Gaillardin C, eds. Molecular Biology and Genetic Engineering of Yeasts[J]. CRC Press, 1992,18: 545-557.
[4] Chien, C.T. ,Bartel, P. L. , Sternglanz, R. & Fields, S. The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Nat. Acad. Sci, 1991, 88: 9578–9582.
[5] M Tanowitz and M von Zastrow. Identification of protein interactions by yeast two-hybrid screening and coimmunoprecipitation[J]. Methods MolBiol, 2004, 259 : 353.
[6] Garabet G. Toby, Erica A. Golemis. Using the Yeast Interaction Trapand Other Two-Hybrid Based Approaches to Study Protein-Protein Interactions [J]. Methods, 2001, 24 (3) : 201.
[7] Hope L. Lancero, Schryl Castaneda. Analysing protein–protein interactions of the Myxococcus xanthus Dif signalling pathway using the yeast two-hybrid system [J]. Microbiology, 2005, 151, 1535–154.
[8] Ilya G. Serebriiskii, Erica A. Golemis. Uses of lac Z to Study GeneFunction: Evaluation ofβ-Galactosidase Assays Employed in the Yeast Two-Hybrid System [J]. Analytical Biochemistry, 2000, 285(1): 1.
[9] Guarente, L.Strategies for the identification of interacting proteins. Proc Natl Acad Sci USA , 1993, 90:1639–1641.
[10] Estojak, J., Brent, R. & Golemis,E. A. Correlation of two-hybrid affnity data with in vitro measurements. Molecular and Cellular Biology, 1995,15: 5820-5829.
[11] Yang M, Wu Z & Fields S. Protein-peptide interactions analyzed with the yeast two-hybrid system. Nucleic Acid Res, 1995,23(7): 1152–1156.
[12] Wang Jun-Jie. Involvement of headless myosin X in the motility of immortalized gonadotropin-releasing hormone neuronal cells. Cell Biology International, 2009: 1-9.
[13] Sousa A D and R E Cheney, Myosin-X: a molecular motor at the cell's fingertips[J]. Trends Cell Biol, 2005, 15(10):533-5399.
[14] Homma k, and M Ikebe. Myosin X is a high duty ratio motor[J]. Biol Chem, 2005, 280: 29381-29391.
[15] Homma,k.,J.Saito,R.Ikebe,andM.Ikebe. Motor function and regulation of myosinX.J.Biol.Chem, 2001,276: 34348- 34354.
[16] Tokuo H and MIkebe, Myosin X transports Mena/VASP to the tip of filopodia [J]. Biochem Biophys Res Commun, 2004, 319(1): 214-220.
[17] Zhang H, Berg J S, Li Z, et al. Myosin-X provides a motor-based link between integrins and the cytoskeleton [J]. Nat Cell Biol, 2004, 6(6): 523-31.
[18] Chavrier P,May the force be with you:Myosin-X in phagocytosis[J]. Nat Cell Biol, 2002, 4(7): E169-71.
[19] Cox D, Berg J S, Cammer M, et al.,Myosin X is a downstream effector of PI(3)K during phagocytosis[J]. Nat Cell Biol, 2002, 4(7): 469-77.
[20] Pi X, Ren R, Kelley R,et al., Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors[J]. J Cell Biol, 2007, 179(7): 1569-82.
[21] Bohil A B, B W Robertson,and R E Cheney,Myosin-X is a molecular motor that functions in filopodiaformation[J].Proc Natl Acad Sci US A, 2006, 103(33): 12411-6.
[22] Bohil A B, B W Robertson,and R E Cheney,Myosin-X is a molecular motor that functions infilopodia for mation[J].Proc Natl Acad Sci USA, 2006, 103(33):12411-6.
[23] Tokuo H, K Mabuchi, and M Ikebe, The motor activity of myosin-X promotes actin fiber convergenceat the cell periphery to initiate filopodia formation[J]. J Cell Biol,2007, 179(2):229-38.
[24] Sarah Woolner, et al., Myosin-10 and actin filaments are essential for mitotic spindle function[J]. J Cell Biol, 2008:1-12.
[25] Toby GG, Gherraby W, Coleman TR, Golemis EA. A novel RING-finger protein, Human Enhancer of Invasion 10 (HEI10), alters mitotic progression through regulation of cyclin B levels[J]. Mol Cell Biol ,2003, 23:2109–2122.
[26] Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY, Kaiser BK, et al. The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases[J]. Trends in Cell Biol, 2000, 10:429–439.
[27] Mine N, Kurose K, Konishi H, et al. Fusion of a sequence from HEI10 (14q11) to the HMGIC gene at 12q15 in a uterine leiomyoma. Jpn[J]. Cancer Res, 2001,92: 135–139.
[28] Gronholm M, Muranen T, Toby GG, et al. A functional association between merlin and HEI10, a cell cycle regulator[J]. Oncogene, 2006.
[29] Evans DG, Sainio M, Baser ME. Neurofibromatosis type 2[J]. J Med Genet, 2000, 37: 897–904.
[30] Xiao GH, Gallagher R, Shetler J, et al. The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1expression[J]. Mol Cell Biol, 2005, 25: 2384–2394.
[31] Mahendra K. Singh, et al. HEI10 negatively regulates cell migration by inhibiting cyclin B/ Cdk1 and other promotility proteins[J]. Oncogene, 2007; 26(33): 4825–4832.
[32] Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments[J]. Cell, 2003, 112(4): 453-465.
[33] Plastino J, Sykes C. The actin slingshot, Curr Opin[J]. Cell Biol, 2005, 17(1): 62-66
[34] Huiwu Zhao, et al. UXT is a novel centrosomal protein essential for cell viability[J]. Molecular Biology of the Cell, 2005, 16: 5857–5865.
[35]Anderson S J, Wilkinson C J , Mayor T, et al. Proteomiccharacterization of the human centrosome by protein correlation profiling[J]. Nature, 2003,426,570–574.
[36] Schroer A, Schneider S, Ropers H & Nothwang HG. Cloning and characterization of UXT, a novel gene inhumanXp11,which is widely and abundantly expressed in tumortissue[J]. Genomics, 1999, 56, 340–343.
[37] R. McGilvray et al. UXT interacts with the transcriptional repressor protein EVI1 and suppresses cell transformation[J]. FEBS, 2007, 274 ,3960–3971.