利用酵母双杂交筛选随机多肽文库的方法研究AP3D1蛋白Ear结构域的结合特性
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摘要
细胞内的膜转运系统牵涉到多个层次的调控过程,包括货物分选、膜泡形成、膜泡运输及融合等。衔接蛋白Adaptin(AP)复合物是膜转运系统中的一种关键调控因子,可以识别目标蛋白并把其分选到运输膜泡内作为货物进行输送。现已发现五种衔接蛋白复合物(AP1-AP5),其中AP3(adaptor protein complex3)主要参与从高尔基体／内体到溶酶体的转运工作。AP3由四个蛋白亚基组成,其中AP3D1的C末端含有一个非常保守的Ear结构域,它可以识别结合多种辅助蛋白形成运输膜泡,但目前该结构域的分子识别机制及由其所介导的生物学调控功能还不清楚。
蛋白质是生命现象的物质基础,而蛋白-蛋白之间的相互作用在生命活动中发挥着极其重要的作用,因此对它的研究可以了解蛋白质在生物体中的作用方式,从而揭示它在生物体内行使的功能。为了能够只通过一次筛选就可以找出Ear结构域的所有配体蛋白,在本论文中,我们利用酵母双杂交技术筛选随机多肽文库的方法,首先找出Ear结构域的结合多肽,然后通过生物信息学分析找出其结合规律并在蛋白质组水平上预测其所有的潜在结合蛋白,最后再通过实验验证新的配体蛋白。
在众多的研究方法中,酵母双杂交系统不仅可以研究活细胞体内的蛋白质相互作用、灵敏度高,而且不需对目的蛋白进行纯化,使实验操作简单易行,因此是研究蛋白质相互作用的一种强有力的方法。另外,我们筛选随机多肽文库,可以尽可能得到所有可能与目的蛋白AP3D1(AP3, deta1subunit)的Ear结构域结合的随机多肽序列,大大提高了文库筛选的效率。
Membrane trafficking in eukaryotic cells is exquisitely regulated at multiple levels, including the regulation of cargo sorting, vesicle formation, trafficking and fusion processes. Adaptor/Adaptin protein (AP) complexes are one of the key regulators of intracellular membrane trafficking, which can dictate the sorting of specific cargoes into transport vesicles for conveying from one membrane compartment of the cell to another. Among the five distinct AP complexes, AP1-AP5, identified in most eukaryotes, AP3complex is largely responsible for the cargo sorting and transport from Golgi/endosomes to lysosomes. AP3D1, one component of the AP3complex, contains a highly conserved Ear domain at the C-terminus, which plays a critical role in the recruitment of multiple accessory proteins to membrane coats. However, it remains unclear how this Ear domain can specifically recognize its target proteins for the sorting and transport.
Proteins perform most of functions in life. And no protein works by itself alone. Therefore, protein-protein interaction plays extremely important role. Study of entire protein interaction networks or protein interactome becomes essential for us to understand how the proteins function in cells. In order to screen all the Ear binding motifs for a particular Ear domain from a single screening, we use the Yeast Two-Hybrid System to investigate the ligand-binding characteristics of the targeted Ear domain by screening random peptide library in this experiment. With these ligand-binding characteristics the potential native binding proteins of these domains in proteomic scale base have been predicted by bioinformatics technology, then find the new ligandins via experimental verification.
However, the Yeast Two-Hybrid System is the most powerful technique in many methods to investigate the protein interactome, because the Y2H system enables highly sensitive detection of protein-protein interactions in vivo without purifying any protein molecules and antibodies, which takes months to prepare. In addition, we use random peptide library to find all the potential Ear binding C terminal sequence can reduce the workload of screening many cDNA libraries, and increase the efficiency.
蛋白质是生命现象的物质基础,而蛋白-蛋白之间的相互作用在生命活动中发挥着极其重要的作用,因此对它的研究可以了解蛋白质在生物体中的作用方式,从而揭示它在生物体内行使的功能。为了能够只通过一次筛选就可以找出Ear结构域的所有配体蛋白,在本论文中,我们利用酵母双杂交技术筛选随机多肽文库的方法,首先找出Ear结构域的结合多肽,然后通过生物信息学分析找出其结合规律并在蛋白质组水平上预测其所有的潜在结合蛋白,最后再通过实验验证新的配体蛋白。
在众多的研究方法中,酵母双杂交系统不仅可以研究活细胞体内的蛋白质相互作用、灵敏度高,而且不需对目的蛋白进行纯化,使实验操作简单易行,因此是研究蛋白质相互作用的一种强有力的方法。另外,我们筛选随机多肽文库,可以尽可能得到所有可能与目的蛋白AP3D1(AP3, deta1subunit)的Ear结构域结合的随机多肽序列,大大提高了文库筛选的效率。
Membrane trafficking in eukaryotic cells is exquisitely regulated at multiple levels, including the regulation of cargo sorting, vesicle formation, trafficking and fusion processes. Adaptor/Adaptin protein (AP) complexes are one of the key regulators of intracellular membrane trafficking, which can dictate the sorting of specific cargoes into transport vesicles for conveying from one membrane compartment of the cell to another. Among the five distinct AP complexes, AP1-AP5, identified in most eukaryotes, AP3complex is largely responsible for the cargo sorting and transport from Golgi/endosomes to lysosomes. AP3D1, one component of the AP3complex, contains a highly conserved Ear domain at the C-terminus, which plays a critical role in the recruitment of multiple accessory proteins to membrane coats. However, it remains unclear how this Ear domain can specifically recognize its target proteins for the sorting and transport.
Proteins perform most of functions in life. And no protein works by itself alone. Therefore, protein-protein interaction plays extremely important role. Study of entire protein interaction networks or protein interactome becomes essential for us to understand how the proteins function in cells. In order to screen all the Ear binding motifs for a particular Ear domain from a single screening, we use the Yeast Two-Hybrid System to investigate the ligand-binding characteristics of the targeted Ear domain by screening random peptide library in this experiment. With these ligand-binding characteristics the potential native binding proteins of these domains in proteomic scale base have been predicted by bioinformatics technology, then find the new ligandins via experimental verification.
However, the Yeast Two-Hybrid System is the most powerful technique in many methods to investigate the protein interactome, because the Y2H system enables highly sensitive detection of protein-protein interactions in vivo without purifying any protein molecules and antibodies, which takes months to prepare. In addition, we use random peptide library to find all the potential Ear binding C terminal sequence can reduce the workload of screening many cDNA libraries, and increase the efficiency.
引文
[1]McNiven MA, Thompson HM. Vesicle formation at the plasma membrane and trans-Golgi network:the same but different [J]. Science.2006,15:313 (5793):1591-4
[2]Blackstone C, O'Kane CJ, Reid E.Hereditary spastic paraplegias:membrane traffic and the motor pathway [J].Nat Rev Neurosci.2011 Jan; 12(1):31-42
[3]Mellman I, Warren G.The road taken:past and future foundations of membrane traffic [J]. Cell.2000 Jan 7; 100 (1):99-112
[4]翟中和,王喜忠,丁明孝.细胞生物学.第3版[M].北京：高等教育出版社,2007.205-207
[5]Drake MT, Zhu Y, Kornfeld S. The assembly of AP-3 adaptor complex-containing clathrin-coated vesicles on synthetic liposomes[J].Mo/Biol Cell.2000 Nov. 11(11):3723-36.
[6]Stoorvogel W, Oorschot V, Geuze HJ. A novel class of clathrin-coated vesicles budding fromendosomes [J]. J Cell Biol.1996 Jan; 132 (1-2):21-33.
[7]Zhu Y, Drake MT, Kornfeld S. Adaptor protein 1-dependent clathrin coat assembly on synthetic liposomes and Golgi membranes [J]. Methods Enzymol.2001;329:379-87
[8]Smythe E. Clathrin-coated vesicle formation:a paradigm for coated-vesicle formation [J]. Biochem Soc Trans.2003 Jun; 31 (Pt 3):736-9. Review
[9]伍祚斌.拟南芥Rab1及Rab2家族基因的克隆和功能研究[D].[硕士学位论文].海南大学,2009
[10]Bonifacino, J.S.and Glick, B.S. The mechanisms of vesicle budding and fusion. Cell,2004,116(2):153-166
[11]Boehm M, Aguilar RC, Bonifacino JS. Functional and physical interactions of the adaptor protein complex AP-4 with ADP-ribosylation factors (ARFs). EMBO J.2001 Nov 15;20(22):6265-76
[12]Boehm M, Bonifacino JS. Genetic analyses of adaptin function from yeast to mammal's [J]. Gene.2002 Mar 20; 286 (2):175-86
[13]Hirst J, Robinson MS.Clathrin and adaptors [J]. Biochim Biophys Acta..1998 Aug 14:1410(1-2):173-93
[14]Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J. Role of API and Gadkin in the traffic of secretory endo-lysosomes [J]. Mol Biol Cell.2011 Jun 15; 22(12):2068-82
[15]Rodionov DG, Honing S, Silye A,Kongsvik TL, von Figura K, Bakke O. Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3 [J]. JBiol Chem.2002 Dec6; 277 (49):47436-43
[16]Cowles CR, Odorizzi G, Payne GS, Emr SD. The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole [J]. Cell,1997. Oct; 3:91(1):109-18
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[18]Dell'Angelica EC, Ooi CE, Bonifacino JS. Beta3A-adaptin, a subunit of the adaptor-like complex AP-3 [J]. JBiol Chem.1997 Jun 13; 272(24):15078-84
[19]Le Borgne R, Alconada A, Bauer U, Hoflack B. The mammalian AP-3 adaptor-like complex mediates the intracellular transport of lysosomal membrane glycoproteins [J]. J.Cell Biol,1998. Nov 6:273(45):29451-61
[20]Kent HM, Evans PR, Schafer IB. Structural basis of the intracellular sorting of the SNARE VAMP7 by the AP3 adaptor complex [J]. Dev Cell,2012; 22(5):979-88
[21]Jones ML, Murden SL, Brooks C, Maloney V. Disruption of AP3B1 by a chromosome 5 inversion:a new disease mechanism in Hermansky-Pudlak syndrome type 2. BMC Med Genet.2013 Apr 4; 14(1):42.
[22]Burgos PV, Mardones GA, Rojas AL, daSilva LL, Prabhu Y, et al. Sorting of the Alzheimer's disease amyloid precursor protein mediated by the AP-4 [J]. Dev Cell,2010, 18:425-436
[23]Heuser JE, Keen J. Deep-etch visualization of proteins involved in clathrin assembly [J]. J Cell Biol.1988.107(3):877-86
[24]Robinson MS, Bonifacino JS Adaptor-related proteins [J]. Curr Opin Cell Biol, 2001,13:444-453
[25]Knuehl C, Brodsky FM. The long and short of adaptor appendages [J]. Nat Struct Biol.2003.10 (8):580-2
[26]Bartel,P.L., C.-T.Chien, R.Sternglanz, S.Fields. Elimination of false positives that arise in using the two-hybrid system. Biotechniques.1993.14:p.920-4
[27]Fields S. The two-hybrid system to detect protein-protein interactions. METHODS'.A Companion to Meth Enzymol,1993,5 (02)
[28]Fields S.and R.Sternglanz. The two-hybrid system:an assay for protein-protein interactions. Trends Genet,1994.286-92
[29]Heslot,H.and C.Gaillardin.. Moleculat Biology and Genetic Engineering of Yeasts. CRC Press.Inc.1992
[30]Hope,I.A. and K.Struhl.. Functional dissection of a eukaryotic transcriptional protein, GNN4 of yeast Cell,1986.46:p.885-94
[31]Keegan,L. and G.Gill, Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science,1986.231:p.699-704
[32]Ma,J.and M.Ptashne.. A new class of yeast transcriptional activators. Cell 1987.51:p.113
[33]Brent,R.and M.Ptashne.. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell,1985.43:p.729-36
[34]Ma,J.and M.Ptashne. Converting a eukaryotic transcriptional inhibitor into an activator. Cell,1988.55:p.443-446
[35]Guarente,L.. Strategies for the identification of interacting proteins. Proc.Natl.Acad.Sci. USA,1993.90:p.1639-1641.
[36]张开慧.酵母双杂交系统及应用[J].价值工程,2011,30(23)
[37]张迪,霍克克等.酵母双杂交技术研究进展[J].高技术通讯,2000,10(3)
[38]刘佳.限制性显示技术在HIV随机多肽文库构建中的应用[D].[硕士学位论文].第一军医大学,2005
[39]黄海明.蛋白质相互作用结构域PDZ结合特性的研究方法[D].[博士学位论文].中国协和医科大学,2004
[40]宋婀莉.高效研究多肽结合结构域结合特性新策略的建立及其应用[D].[博士学位论文].中国协和医科大学,2007
[41]Schmid EM, Ford MG. Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly. PLo S Biol.2006 Sep; 4(9):e262.
[2]Blackstone C, O'Kane CJ, Reid E.Hereditary spastic paraplegias:membrane traffic and the motor pathway [J].Nat Rev Neurosci.2011 Jan; 12(1):31-42
[3]Mellman I, Warren G.The road taken:past and future foundations of membrane traffic [J]. Cell.2000 Jan 7; 100 (1):99-112
[4]翟中和,王喜忠,丁明孝.细胞生物学.第3版[M].北京：高等教育出版社,2007.205-207
[5]Drake MT, Zhu Y, Kornfeld S. The assembly of AP-3 adaptor complex-containing clathrin-coated vesicles on synthetic liposomes[J].Mo/Biol Cell.2000 Nov. 11(11):3723-36.
[6]Stoorvogel W, Oorschot V, Geuze HJ. A novel class of clathrin-coated vesicles budding fromendosomes [J]. J Cell Biol.1996 Jan; 132 (1-2):21-33.
[7]Zhu Y, Drake MT, Kornfeld S. Adaptor protein 1-dependent clathrin coat assembly on synthetic liposomes and Golgi membranes [J]. Methods Enzymol.2001;329:379-87
[8]Smythe E. Clathrin-coated vesicle formation:a paradigm for coated-vesicle formation [J]. Biochem Soc Trans.2003 Jun; 31 (Pt 3):736-9. Review
[9]伍祚斌.拟南芥Rab1及Rab2家族基因的克隆和功能研究[D].[硕士学位论文].海南大学,2009
[10]Bonifacino, J.S.and Glick, B.S. The mechanisms of vesicle budding and fusion. Cell,2004,116(2):153-166
[11]Boehm M, Aguilar RC, Bonifacino JS. Functional and physical interactions of the adaptor protein complex AP-4 with ADP-ribosylation factors (ARFs). EMBO J.2001 Nov 15;20(22):6265-76
[12]Boehm M, Bonifacino JS. Genetic analyses of adaptin function from yeast to mammal's [J]. Gene.2002 Mar 20; 286 (2):175-86
[13]Hirst J, Robinson MS.Clathrin and adaptors [J]. Biochim Biophys Acta..1998 Aug 14:1410(1-2):173-93
[14]Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J. Role of API and Gadkin in the traffic of secretory endo-lysosomes [J]. Mol Biol Cell.2011 Jun 15; 22(12):2068-82
[15]Rodionov DG, Honing S, Silye A,Kongsvik TL, von Figura K, Bakke O. Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3 [J]. JBiol Chem.2002 Dec6; 277 (49):47436-43
[16]Cowles CR, Odorizzi G, Payne GS, Emr SD. The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole [J]. Cell,1997. Oct; 3:91(1):109-18
[17]Dell'Angelica EC, Ohno H, Ooi CE, Rabinovich E, Roche KW, Bonifacino JS.AP-3:an adaptor-like protein complex with ubiquitous expression [J]. EMBO J.1997 Mar 3; 16(5):917-28
[18]Dell'Angelica EC, Ooi CE, Bonifacino JS. Beta3A-adaptin, a subunit of the adaptor-like complex AP-3 [J]. JBiol Chem.1997 Jun 13; 272(24):15078-84
[19]Le Borgne R, Alconada A, Bauer U, Hoflack B. The mammalian AP-3 adaptor-like complex mediates the intracellular transport of lysosomal membrane glycoproteins [J]. J.Cell Biol,1998. Nov 6:273(45):29451-61
[20]Kent HM, Evans PR, Schafer IB. Structural basis of the intracellular sorting of the SNARE VAMP7 by the AP3 adaptor complex [J]. Dev Cell,2012; 22(5):979-88
[21]Jones ML, Murden SL, Brooks C, Maloney V. Disruption of AP3B1 by a chromosome 5 inversion:a new disease mechanism in Hermansky-Pudlak syndrome type 2. BMC Med Genet.2013 Apr 4; 14(1):42.
[22]Burgos PV, Mardones GA, Rojas AL, daSilva LL, Prabhu Y, et al. Sorting of the Alzheimer's disease amyloid precursor protein mediated by the AP-4 [J]. Dev Cell,2010, 18:425-436
[23]Heuser JE, Keen J. Deep-etch visualization of proteins involved in clathrin assembly [J]. J Cell Biol.1988.107(3):877-86
[24]Robinson MS, Bonifacino JS Adaptor-related proteins [J]. Curr Opin Cell Biol, 2001,13:444-453
[25]Knuehl C, Brodsky FM. The long and short of adaptor appendages [J]. Nat Struct Biol.2003.10 (8):580-2
[26]Bartel,P.L., C.-T.Chien, R.Sternglanz, S.Fields. Elimination of false positives that arise in using the two-hybrid system. Biotechniques.1993.14:p.920-4
[27]Fields S. The two-hybrid system to detect protein-protein interactions. METHODS'.A Companion to Meth Enzymol,1993,5 (02)
[28]Fields S.and R.Sternglanz. The two-hybrid system:an assay for protein-protein interactions. Trends Genet,1994.286-92
[29]Heslot,H.and C.Gaillardin.. Moleculat Biology and Genetic Engineering of Yeasts. CRC Press.Inc.1992
[30]Hope,I.A. and K.Struhl.. Functional dissection of a eukaryotic transcriptional protein, GNN4 of yeast Cell,1986.46:p.885-94
[31]Keegan,L. and G.Gill, Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science,1986.231:p.699-704
[32]Ma,J.and M.Ptashne.. A new class of yeast transcriptional activators. Cell 1987.51:p.113
[33]Brent,R.and M.Ptashne.. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell,1985.43:p.729-36
[34]Ma,J.and M.Ptashne. Converting a eukaryotic transcriptional inhibitor into an activator. Cell,1988.55:p.443-446
[35]Guarente,L.. Strategies for the identification of interacting proteins. Proc.Natl.Acad.Sci. USA,1993.90:p.1639-1641.
[36]张开慧.酵母双杂交系统及应用[J].价值工程,2011,30(23)
[37]张迪,霍克克等.酵母双杂交技术研究进展[J].高技术通讯,2000,10(3)
[38]刘佳.限制性显示技术在HIV随机多肽文库构建中的应用[D].[硕士学位论文].第一军医大学,2005
[39]黄海明.蛋白质相互作用结构域PDZ结合特性的研究方法[D].[博士学位论文].中国协和医科大学,2004
[40]宋婀莉.高效研究多肽结合结构域结合特性新策略的建立及其应用[D].[博士学位论文].中国协和医科大学,2007
[41]Schmid EM, Ford MG. Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly. PLo S Biol.2006 Sep; 4(9):e262.