BTB基因家族成员BTBD8、KLHL7和KLHL9的克隆和功能研究

【作者】 徐坚；

【导师】 谢毅；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 BTB结构域和kelch结构域都是在进化上高度保守的结构域,广泛存在于从酵母到人类的各种物种中。在人类基因组中发现了很多BTB/kelch蛋白,提示这类蛋白功能的重要性和多样性。现在越来越多的实验证据证明BTB/kelch蛋白在胞质分裂、细胞骨架功能维持、细胞周期调控、肿瘤发生、神经发育等重要生理过程中都起了重要的作用。BTB结构域的主要功能是介导蛋白相互作用,它在PLZF和BCL6等转录因子的转录调控和参与Cul3介导的泛素途径中都有重要作用。kelch蛋白是近年来发现的一类新的细胞骨架结合蛋白,可以与actin细胞骨架作用从而对细胞骨架功能进行调控。更多的研究表明kelch结构域的功能多样化,参与了一系列重要的信号传导途径,在细胞周期调控和肿瘤发生中都发挥了重要作用。例如,研究发现NRP/B可以与核骨架结构作用,并且NRP/B受到p53的转录调控,进而影响ERK/MAPK途径和AKT途径。ENC-1可以与非磷酸化的p110Rb蛋白相互作用而影响神经元的细胞周期和分化,在脂肪细胞分化和结肠癌发生中ENC-1也起了重要的作用。对人类Mayven蛋白的最新研究表明Mayven在乳腺癌的发生中起了重要作用,Mayven可以通过激活AP-1、c-jun、cyclin D等的表达对细胞周期进行调控。 我们从本实验室的高质量人胎脑文库中克隆得到了三个含有BTB结构域的新成员:BTBD8、KLHL7和KLHL9,并且KLHL7和KLHL9同时含有kelch-repeat结构域。通过信息学分析发现我们得到的BTBD8、KLHL7和KLHL9的cDNA均为全长形式,它们分别编码含有305个氨基酸、538个氨基酸和617个氨基酸的蛋白,并且都含有保守的BTB结构域和kelch结构域。通过Northern杂交、RT-PCR和原位杂交等实验,我们发现了它们各自特异的表达谱模式:BTBD8在8种胎儿组织中广泛表达而在16种成人组织中表达量都很低,这种表达模式提示BTBD8可能在发育过程中起一定的作用;KLHL7在多种肿瘤组织和细胞系中表达并且在胰腺癌组织中表达量显著高于癌旁组织,提示其在肿瘤发生中可能起了重要作用;KLHL9在胎儿肝组织中表达显著高于成人肝组织,提示在胎儿肝组织中行使一些重要功能。对这三个基因的细胞定位研究发现BTBD8在AD293细胞中为细胞核定位而在NIH3T3细胞中为全细胞定位,提示在这两种细胞系中可能有不同的功能。而对KLHL7和KLHL9的定位研究发现它们的BACK结构域对定位都有重要作用。利用免疫共沉淀实验我们确定了KLHL7和KLHL9都能与actin结合,提示KLHL7和KLHL9可能在维持细胞骨架功能和参与骨架信号传导中有重要作用。更多还原

【Abstract】 BTB domain and kelch-repeat domain are both highly conserved domains in evolution, they exist widely in nearly all organisms from yeast to human genome. The fact that there are many BTB/kelch proteins in the human genome suggests important and various roles of such proteins. Now more and more results show that BTB/kelch proteins participate in many vital cell activities, such as cytokinesis, actin cytoskeleton, cell cycle, tumorigenesis, neuronal development and the like. The fundamental roles of BTB domain lie on its ability to mediate protein interactions. Some BTB-domain containing transcription factors, such as PLZF and BCL6 play important roles in regulation of gene transcription. Recent study reveals that BTB domain can interact with Cul3 E3 ligase and take part in ubiquitin pathway. kelch proteins are considered to be a novel family of actin-binding proteins (ABPs), they can interact with actin cytoskeleton so that exert function on stability of cytoskeleton. More results demonstrate that kelch domain may have diverse functions in many signal transduction pathways. NRP/B locates in nuclear cytoskeleton. It regulates ERK/MAPK pathway and AKT pathway via regulation by p53. ENC-1 can interact with nonphosphorylated p110Rb protein and play roles in neuron cell cycle and differentiation. ENC-1 also has roles in adipocyte differentiation and colon carcinoma. Study on Mayven reveals that Mayven plays important roles in breast carcinoma, it can regulate cell cycle by activating expression of AP-1, c-jun and cyclin D.From the high quality human fetal brain library, we cloned three novel genes containing BTB domains. They are designated as BTBD8, KLHL7 and KLHL9 in agreement with HUGO nomenclature committee. KLHL7 and KLHL9 also have kelch-repeat domains in their carboxyl-terminals. Through bioinformatical analysis we showed that the cDNAs we got are full-length ones. The ORFs of BTBD8, KLHL7 and KLHL9 encode proteins of 305 amino acids, 538 amino acids and 617 amino acids, respectively. We studied expression profiles of these three genes by Northern blot, RT-PCR and In situ hybrid. The results showed that BTBD8 was strongly expressed in 8 fetal tissues while weakly in 16 adult tissues, suggesting it might play a role during development. KLHL7 was ubiquitously expressed in several tumor tissues and tumor cell lines, especially its expression was significantly upregulated in pancreatic carcinoma compared with normal pancreatic tissue. All these results suggested KLHL7 might play important roles in tumorigenesis and tumor progress. KLHL9 was also ubiquitously expressed in multiple tissues but it had更多还原