人泛素活化酶UBE1DC1、UBE1及人泛素结合酶UBE2F的功能研究

【作者】 郑眉；

【导师】 毛裕民；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 泛素UB或类泛素分子UBLs作为一种蛋白修饰分子在泛素结合途径的第一步起作用。此途径参与体内许多重要的生理功能,包括细胞周期的调控、抗原递呈、转录调控、凋亡、DNA损伤和修复等。泛素与蛋白底物的结合则是泛素化途径必需的过程,它经由三步级联反应机制进行下去,泛素活化酶作为在泛素化途径第一步起重要作用的蛋白,它能活化泛素或者类泛素分子。泛素活化酶E1在需要ATP的反应中激活泛素UB或者类泛素分子产生一个高能硫羟酸酯媒介,E1-S～泛素。泛素结合酶E2结合活化的泛素或者类泛素分子,并将激活的泛素部分转移到泛素连接酶E3或底物。本文对泛素活化酶UBE1DC1、UBE1及泛素结合酶UBE2F的功能进行了初步探讨。人泛素活化酶UBE1DC1A（ubiquitin-activating enzyme E1-domaincontaining 1）及其新的剪接体UBE1DC1B被认为是类泛素活化酶家族成员。UBE1DC1与人泛素活化酶UBE1的相应区域具有高度同源性,且两者都具有保守的ThiF结构域,含有ATP结合结构域（GXGXXG）。通过大肠杆菌表达获得了UBE1DC1A、UBE1DC1B、UB以及多种类泛素分子UBLs蛋白,经体外免疫印记检测UBE1DC1活化泛素及类泛素分子的特异性发现,它除了能活化类泛素分子Ufm1外还能活化类泛素分子SUMO2,这得到了体内实验结果的验证。通过Blast程序同源性比较发现Ufm1和SUMO2蛋白无同源性,说明UBE1DC1可能不像其他类泛素活化酶只能激活一种类泛素分子,它能同时活化两种不同类型的类泛素分子。同源比较和突变试验确定了Cys250是其活性位点。UBE1DC1和SUMO2在AD293细胞内的定位试验结果表明,当UBE1DC1与SUMO2共转染细胞后,UBE1DC1的细胞定位从细胞质定位变成了细胞核定位,推测UBE1DC1可能在细胞质中激活SUMO2之后转移到细胞核中,进而参与E2反应。UBE1是首个被发现的泛素活化酶,它在泛素化途径的第一步活化泛素上起重要作用。制备获得高纯度的UBE1是研究其蛋白质结构与生化性质以及用于泛素化途径下游相关酶活性测定的基础。在全基因合成的基础上,全长UBE1基因成功地在大肠杆菌中获得了高表达,根据表达载体所携带的6His标签以及UBE1能活化UB形成UB-UBE1复合物的特性,经Ni-NTA柱层析和Strep-TactinSepharose柱层析,纯化得到了高纯度的UBE1蛋白,纯化得到的UBE1蛋白经活性分析发现具有泛素活化酶活性。通过对UBE1、UB以及UB-UBE1复合物的二级结构测定发现,UBE1与UB反应形成UB-UBE1交联复合物后,它的二级构象与单一的UBE1二级构象相比发生了显著变化,说明了UB与UBE1交联后引起了UBE1结构较大的变化,特别是α螺旋的含量明显增加。泛素结合酶则在泛素修饰途径的第二步反应过程中起重要作用。通过筛选cDNA文库,我们从人胎脑文库中克隆到了一种新的人泛素结合酶ubiquitin-conjugating enzyme E2F,将它命名为UBE2F。UBE2F基因cDNA序列全长1366bp,编码一个185aa蛋白。它与大部分的人泛素结合酶一样,具有同源性较高的保守结构域UBCc,且都含有半胱氨酸活性位点、UB硫脂键互作位点及E3互作位点。UBE2F与生物体中同源蛋白是高度保守的,且UBE2F基因在绝大多数人体组织中都有表达,因而我们推测UBE2F应该是具有泛素活化酶活性。在此研究中,我们发现UBE2F能结合被UBE1泛素化的UB,产生UB-UBE2F交联复合物,可见它确实是一种泛素结合酶,并可能在泛素修饰途径中起作用;而将其117位半胱氨酸突变成丙氨酸之后,就不能形成UB-UBE2F交联复合物,可见Cys117是它的活性位点。另外,UBE2F和突变体UBE2F^C117A在AD293细胞中都是全细胞定位,可见Cys117活性位点对其定位无显著影响。更多还原

【Abstract】 Ubiquitin（UB） and Ubiquitin-like proteins（UBLs） as a kind of protein modifications are activated in the first step of ubiquitination pathway.Ubiquitination pathway plays major roles in a variety of basic physiological functions involved in cell cycle,apoptosis,antigen presentation,transcription,DNA damage and repair.The system of ubiquitination conjugation involves 3 steps.The ubiquitin-activating enzyme（E1） plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins.The ATP-dependent ubiquitin-activating enzyme （E1） forms a covalent bond with ubiquitin（UB） or ubiquitin-like proteins（UBLs） in the first step.Activated UB or UBLs were transferred to ubiquitin-conjugating enzyme E2,and then transferred to ubiquitin-protein ligases E3 or substrates by ubiquitin-eonjugating enzyme E2.The function of ubiquitin-activating enzyme E1-domain containing 1（UBE1DC1）,ubiquitin-activating enzyme E1（UBE1） and ubiquitin-conjugating enzyme E2F（UBE2F） were discussed in the paper.In this report,full length of UBE1DC1A and a novel splice variant of full length gene of UBE1DC1A,UBE1DC1B,were cloned and purified.The cDNAs of UBE1DC1A and UBE1DC1B contain an open reading frame encoding 404 amino acids and 348 amino acids respectively with analogical conserved ThiF domain and ATP-binding domain（GXGXXG） by blast analysis.UBE1DC1A,UBE1DC1B,UB and UBLs were expressed in E.coli and purified by chromatography of Ni-NTA His-Bind Superflow.UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2,besides Ufm1,both in vitro and in vivo by immunological analysis.It indicated that UBE1DC1 could activate two different ubiquitin-like proteins,SUMO2 and Ufm1,which have no significant similarity with each other.It was porved that Cys250 of UBE1DC1 was active site by comparing with homologous proteins and mutation analyzing.Subceilular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm;whereas UBE1DC1 was mainly distributed in the nucleus when it was cotransfected with SUMO2.It presumed that UBE1DC1 transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.UBE1 is an ubiquitin-activating enzyme as the same as UBE1DC1.UBE1 as the first member of ubiquitin-activating enzyme plays a key role in the first step of ubiquitin-proteasome pathway to activate ubiquitin.Reaserching the structure and biochemical property of protein,and analyzing the activity of enzymes which were related with ubiquitin-proteasome pathway,are based on obtaining high purity of UBE1 protein.After synthesized full length of UBE1gene,6His-UBE1 was highly expressed in E.coli,and purified by chromatography of Ni-NTA His-Bind Superflow and Strep-Tactin Sepharose,according to 6His-tag of pET28b vector and the characteristics that UBE1 could activate UB to forming UB-UBE1 intermediate complex.We identified that high purity of UBE1 was isolated by the method,and it could activated UB to ubiquitin-conjugating enzyme E2s.On the other hand,the mechanisms how UBE1 activated UB to form UB-UBE1 intermediate complex in tertiary structure is not clearly.Analyzed by circular dichroism spectra（CD）,it found that the conformation of UB-UBE1 intermediated complex was changed visiblily compared with UBE1,and the contents ofα-helix increased especially.The ubiquitin-conjugating enzyme（E2） plays a key role in the second step of ubiquitination pathway to conjugate ubiquitin or ubiquitin-like proteins,and transfers them to ubiquitin-protein ligases E3.The ubiquitin-conjugating enzyme E2F（named UBE2F） was selected through large-scale cDNA cloning and sequencing.The cDNAs of UBE2F contains an open reading frame encoding 185 amino acids with analogical conserved UBCc domain and active site Cys by blast analysis.UBE2F is proved to conjugat UB in vitro by immunological analysis,and the active site was Cys117 because UBE2F^C117A could not conjugat activated-UB.Subcellular localization in AD293 cells revealed that UBE2F was especially distributed in the cytoplasm and nucleus like UBE2F^C117A.It presumed that Cys117 of UBE2F was unrelated with localization when it was mutated to Ala.更多还原