【作者】 钟英丽；

【导师】 张健；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 AP-2是一个功能重要的转录因子家族，在哺乳动物的胚胎发育过程中，其表达受到严格的时空调控，参与多种组织和器官的细胞分化和形态发生，如神经脊，颅面部，四肢，躯干部，心脏和肾脏的发育。已经被克隆和研究的AP-2家族成员有AP-2α，AP-2β，AP-2γ，AP-2δ与AP-2ε，都能以同源或异源二聚体的形式结合于保守的DNA序列5′-GCCNNNGGC-3′。AP-2α是AP-2家族中最早被克隆和研究最多的一个成员。其功能不仅表现为胚胎发育中的重要调控子，还通过转录调控下游靶基因参与多个生理过程的调节，包括细胞生长，分化，癌变和凋亡等。作为一个多功能的调控因子，AP-2α有多个协同作用的蛋白伙伴。为了找到更多的与AP-2α相互作用的蛋白因子，我们用全长的AP-2α作为诱饵蛋白筛选了HeLa cDNA文库。得到了一个阳性克隆为人类的TESTIN(TES)基因，另一个为actinin(ACTN)基因。两者都是细胞骨架相关的蛋白，都能与actin相互作用。其中TES具有PET-LIM结构，在其C端有先后排列的3个LIM domain，可能与癌症的抑制相关。在本论文中，我们重点研究了TES与AP-2α．的相互作用以及TES的功能。我们表达纯化了全长和多种分段的AP-2α与TES蛋白，并制备了相应的抗血清。AP-2α与TES蛋白的相互作用通过体外pull-down与细胞裂解液中的免疫共沉淀实验得到了进一步验证。我们用体外pull-down实验将两者的相互作用定位于两个蛋白的C端。另外，我们还检测了TES蛋白的分子内相互作用，发现其N端通过C端的LIM3在体外相互结合。在荧光素酶报告系统中，我们的结果表明TES的过表达能够抑制AP-2α对ERBB2启动子的转录活性，而且除了全长，TES的C端与N端似乎都有一定的作用。在细胞免疫荧光化学分析中，我们发现本底TES蛋白定位有多种模式，特别是能位于内质网和细胞核中。我们的结果显示TES与AP-2α的表达在细胞核中有部分重叠区域。有趣的是，我们还发现TES过表达引起本底的AP-2α表达水平降低。而对TES进行RNA干扰可以大大提高本底ERBB2基因的转录水平。总的来说，我们的研究表明AP-2α与TES存在着体外相互作用，在细胞内可能处于同一复合体中，两者的相互作用可能对AP-2α介导的ERBB2转录激活起到抑制。同时我们的结果也为抑制癌症的功能提供了新的机制-通过抑制AP-2α的转录活性而起作用。而TES蛋白新的细胞内定位表明其功能不仅与细胞骨架相关，还在细胞核等区域内有一定的作用，AP-2α就是TES在细胞核中第一个相互作用的协助伙伴。TES在细胞核中的具体功能将有待进一步深入研究和发现。更多还原

【Abstract】 AP-2 is a critical transcriptional family temporally and spatially regulated in mammal embryo development including neural crest, cranial face, limbs, heart and kidney. To date, five members of the AP-2 family of transcription factors, AP-2α, AP-2β, AP-2γ, AP-2δand AP-2εhave been identified and all of them can bind as homo- or hetero-dimers to the typical consensus sequence of 5’-GCCNNNGGC-3’. AP-2αis the first gene cloned and the relatively best characterized among the AP-2 gene family. It has been shown that AP-2αis an important regulator that mediates essential events in embryo development and also participate in many biological processes such as cell growth, differentiation, carcinogenesis, or apoptosis through the transcriptional control of multiple target downstream genes. To be a functional regulator, AP-2αis co-operator with a number of partners. To identify more of the interacting co-activators of AP-2α, a HeLa eDNA library was screened by yeast two-hybrid assay using the full length sequence of AP-2αas a bait protein. One of the positive clones was identified by the yeast two-hybrid assay as the human TESTIN （TES） gene and the other was actinin （ACTN）. Both proteins can bind with the actin involving in the cytoskeleton. The TES has a PET domain in the NH₂-terminus and 3 tandem LIM domains in the COOH-terminus, which has been reported as a candidate of tumor suppressor. In this study, we put emphasis to the TES function research and the relevance between TES AP-2α. The full-length or truncations of AP-2αand TES proteins were expressed and purified, and then corresponding antiserums are prepeared. The interaction between TES and AP-2αproteins was confirmed by pull-down assay in vitro and co-immunoprecipitation in vivo. Furthermore, we found that the interaction domains between TES and AP-2αlie in the COOH-termini of both proteins. In addition, we also detected the intramolecular interaction between the NH₂- and COOH-terminal of the TES proteins, and found the detailed region responsible for binding with the NH₂-terminus was LIM3 in the COOH-terminus of TES. In the luciferase reporter analysis, overexpression of TES suppressed the transcriptional activity of AP-2αover the promoter of ERBB2 gene. It is a puzzle that the two termini of TES also had some function upon the transcriptional activity of AP-2α. In the cellular immunofluorescence assays, we showed a variable endogenous TES localization especially in the nucleus and ER. Further, we demonstrated that TES and AP-2αwere partially co-localized in thenucleus. Moreover, it is interesting that the TES overexpression leads to a decreased level of AP-2αprotein. While transfection of siRNA specifically against TES gene resulted in a significant increase of ERBB2 transcription. Taken together, our studies revealed that AP-2αis physically associated with TES in vitro and formed a complex with TES in vivo. And their interaction may be involving an inhibitory mechanism over AP-2-mediated transactivation of ERBB2 gene. At the same time, we also suggest a new role of TES as a tumor suppressor by suppressing the transcriptional activity of AP-2α. Whereas the novel cellular localization of TES protein implicates that TES functions more than a cytoskeleton associated protein, and its first physical and functional partner in nucleus may be AP-2α.更多还原