节点文献
两个新的蛋白质结构域的鉴定暨生长分化因子3功能的初步探讨
【作者】 刘湘华；
【导师】 余龙；
【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士
【摘要】 本论文分为2个部分，第一部分是蛋白质结构域的鉴定，第二部分是关于GDF3的初步功能分析以及外源重组表达的研究。在第一部分中，通过生物信息学的方法鉴定了两个新的结构域。NCD3G[GPCRs家族3中的9个半胱氨酸结构域]，是一个在GPCRs(G蛋白耦联受体)家族3中鉴定到的新蛋白质结构域。它存在于除γ-氨基丁酸转氨酶受体以外的所有GPCRs家族3的成员(包括亲代谢性谷氨酸盐受体，钙敏受体，信息素受体和味觉受体)蛋白中。序列分析显示NCD3G含有9个高度保守的半胱氨酸残基，形成4个β折叠和3对二硫键。通过同源模建，我们获得了NCD3G的结构模型，它显示该结构域包含两个重复的A1模体，并提示NCD3G很可能在GPCRs跨膜信号传导过程中起到非常重要的作用，而且它位于细胞膜外，可成为潜在的药物作用靶点。CARDP是我们在多种细菌、古生菌的表面蛋白、膜蛋白和分泌蛋白中发现的一种新的蛋白质结构域，它包含有多个高度保守的甘氨酸、亮氨酸、酸性氨基酸残基和酰胺残基，具有六个β折叠区。CARDP可能在细胞基质粘附和底物结合的过程中发挥着重要作用。在第二部分中，我们运用RT-PCR，原位杂交和免疫组化等技术分析了GDF3在正常组织，肿瘤细胞株及肝癌样本，以及3T3-L1分化过程中的表达情况。结果表明GDF3在大鼠海马组织的各区中表达有差异，在小脑的浦肯野氏细胞中有大量表达。对16例人的肝癌样本的检测表明，癌组织中GDF3表达有上调趋势，特别在大多数第三期癌症样本中GDF3高表达。对15种细胞株的检测表明，肿瘤细胞株中的GDF3表达明显增加。我们检测了前脂肪细胞的分化过程中GDF3的表达，结果显示GDF3在处于分化过程中的前脂肪细胞中高表达，在分化前的前脂肪细胞和分化完成后的脂肪细胞中没有表达，我们的结果提示了GDF3对于中枢神经系统、肿瘤和脂肪细胞分化可能有重要的作用，但其具体机制需进一步研究。
【Abstract】 The dissertation consists of two parts: the first part describes the identification of NCD3G and CARDP, two novel protein domains; the second part describes the expression of GDF3 in brain, tumor cells and 3T3-L1, and the reconstruction of prokaryotic and eukaryotic vector for GDF3.In the first part, we identified two novel domains using bioinformatic tools. NCD3G is conserved in family 3 GPCRs, including metabotropic glutamate receptors, calcium sensing receptors, pheromone receptors and taste receptors, with the exception of GABAB receptors. Sequence analysis indicates that NCD3G domain contains nine highly conserved cysteine residues, four β strands and three disulfide bridges. Structural modeling suggests that NCD3G maybe consists of two A1 repeats and the conserved residues are related with certain familial diseases. NCD3G may play important role in the signal transduction, and it may be a target for the development of new drug candidates. CARDP is a novel domain shared by various bacterial and archael surface-, membrane-and secreted proteins with several highly conserved glycine, leucine, acidic residues and acylamino acids. Several lines of evidence suggest that the CARDP domain may have essential functions in cell-matrix adhesion and substance binding.In the second part, we analyzed the expression of GDF3 in some tissues and cell lines by RT-PCR, in situ hybridization and immunostaining on the mRNA and protein level. GDF3 is differently distributed in hippocampus CA2 and CA1 region, and it is expressed highly in Purkinje cells in cerebellum. The expression of GDF3 is upregulated in 16 liver cancer tissues, especially in the third phase of liver cancer patients. GDF3 is highly expressed in the tumor cell lines. During the differentiation of 3T3-L1, the expression of GDF3 changed significantly. Our results suggest that GDF3 may play important roles in the central nervous system, tumorogenesis and the differentiation of3T3-L1, and it needs further study. In order to obtain active GDF3, we constructed prokaryotic and eukaryotic vectors to express recombinant GDF3 in vitro, it will be helpful for the function study and application of GDF3.