【作者】 赵为；

【导师】 红凌；

【作者基本信息】 华中科技大学 ， 遗传学， 2010， 硕士

【摘要】 异戊烯化转移酶一类是调节细胞信号转导网络的重要蛋白分子,它可以影响细胞的癌变。在蛋白质翻译后,异戊烯化转移酶通过硫脂键(-C-S-C)将异戊稀基团转移到蛋白质C端的半胱氨酸残端上,使被修饰蛋白能附着于细胞膜脂上。真核生物细胞内很多蛋白质需要经过异戊烯化才能促进细胞生长分化,维持细胞形态活力和细胞分裂周期等。大部分被异戊烯化转移酶所修饰的蛋白属于Ras家族蛋白,如Ras,Rab等。TERE1/UBIAD1蛋白一类是人体中新的异戊烯转移酶,已经被证明在人体MK-4的合成过程中起着重要的作用。TERE1/UBIAD1与人体膀胱癌、前列腺癌等多种癌症相关,并且TERE1/UBIAD1的突变导致施奈德结晶状角膜营养不良疾病,表现为系统脂质代谢紊乱而引起脂质在眼角膜的异常堆积。人体TERE1/UBIAD1基因和果蝇heix基因同源。在果蝇中,heix是一个肿瘤抑制基因,控制和调节着果蝇血细胞的增殖和分化。目前,对TERE1/UBIAD1的研究逐渐成为肿瘤相关研究领域关注的热点。本文采用生物信息学、分子生物学、细胞生物学和荧光成像等手段研究了TERE1/UBIAD1的亚细胞定位,跨膜结构和分子进化;预测和鉴定果蝇heix基因的启动子,以及调节heix基因表达的microRNAs。生物信息学研究结果表明TERE1/UBIAD1蛋白有多个疏水区域,TERE1/UBIAD1是一个八次跨膜的膜蛋白。TERE1/UBIAD1蛋白定位在细胞膜,内质网和高尔基体上面。分子进化分析表明TERE1/UBIAD1是一类异戊烯转移酶或者甲基萘醌合成酶。TERE1/UBIAD1蛋白在空间上面是一个有八个跨膜区段围成的桶状结构。活细胞荧光成像结果表明TERE1/UBIAD1与细胞内质网和高尔基体共定位,并且少量的TERE1/UBIAD1蛋白定位在除细胞膜,内质网和高尔基体之外的细胞器上,TERE1/UBIAD1在高尔基体上面的分布比较多。免疫荧光结果表明TERE1/UBIAD1的N端游离在细胞的胞浆中,并且其Loop1结构比较靠近细胞膜内侧。TERE1/UBIAD1截短体实验表明TERE1/UBIAD1的N端对于TERE1/UBIAD1的亚细胞定位起着重要的作用。本研究成功的克隆了人体Hras基因,并将蓝色荧光蛋白BFP基因连接到Hras基因的5`端,构建了pBFP-hras重组质粒,能在真核细胞中表达出BFP和Hras的融合蛋白,为研究TERE1/UBIAD1与Hras之间的关系提供了很好的帮助。果蝇heix基因调控研究结果表明,果蝇heix基因的启动子位于转录起始位点上游170bp和下游350bp之间。果蝇heix基因的启动子包含多个转录调控元件,并且具有较高的转录活性。果蝇heix基因的3`UTR区域存在特定microRNA的结合靶位点,heix基因的表达可能受到果蝇多个microRNAs转录后水平的调控,如dmiRNA3,dmiRNA309,dmiRNA318,dmiRNA4,dmiRNA133和dmiRNA79。该研究成果为进一步研究果蝇heix基因的功能及其表达调控奠定了基础。更多还原

【Abstract】 Prenyltransferase is a class of important protein regulating intracellular signal transduction network. It has profound impact on cancer cells. Following protein synthesis, prenyltransferase transfers the isopentene radical to the cysteine residue in the C terminal of protein. So the modified protein can attach to the cell membrane. Many proteins of eukaryotic cells need prenylation to promote cell growth and differentiation, and to maintain cell morphology and cell division cycle. The most proteins modified by prenyltransferase are Ras family proteins, such as Ras, Rab.Human TERE1/UBIAD1 protein is a new class of prenyltransferase. It has been proven that TERE1/UBIAD1 is a novel human MK-4 biosynthetic enzyme, which is related to human bladder cancer, prostate cancer and other cancers. TERE1/UBIAD1 is a candidate gene for Schnyder crystalline corneal dystrophy (SCCD) disease, an autosomal dominant disorder involving deposition of cholesterol in the corneal stroma. Drosophila heix and human TERE1/UBIAD1 are homologous genes. In Drosophila, heix is a tumor suppressor gene. It regulates the proliferation and differentiation of Drosophila blood cells. At present, the research on the TERE1/UBIAD1 has gradually become one of the focuses in the cancer research field. In this paper, we have studied the subcellular localization, transmembrane structure and molecular evolution of TERE1/UBIAD1 using bioinformatics, molecular biology, cell biology and fluorescence imaging methods. Meanwhile we have predicted and identified the activity of Drosophila heix gene promoter, and the microRNAs regulating the expression of heix.Our bioinformatics results indicate that TERE1/UBIAD1 protein has multiple hydrophobic regions. TERE1/UBIAD1 is an eight transmembrane protein located on the cell membrane, endoplasmic reticulum and Golgi apparatus. Phylogenetic analysis showed that TERE1/UBIAD1 is a class of prenyltransferase or menaquinone synthase. TERE1/UBIAD1 protein has a barrel structure surrounded by eight transmembrane segments.Live cell fluorescence imaging results indicate that TERE1/UBIAD1 is co-localized with endoplasmic reticulum and Golgi apparatus. Also a small amount of TERE1/UBIAD1 protein is located in the other organelles besides the cell membrane, endoplasmic reticulum and Golgi apparatus. The main distribution of TERE1/UBIAD1 is on the Golgi apparatus. The immunofluorescence result showed that the N-terminal of TERE1/UBIAD1 resides in the cell cytoplasm, and its Loop1 structure is closer to the inner membrane. The truncated TERE1/UBIAD1 experiments indicate that the N-terminal of TERE1/UBIAD1 plays an important role in the subcellular localization of TERE1/UBIAD1.The human Hras gene has been successfully cloned. And the blue fluorescent protein(BFP) gene has been connected to the 5` end of Hras gene, to construct pBFP-hras DNA recombinant plasmid, which could express the fusion protein BFP-hras in eukaryotic cell. These have provided solid grounds for the study of the relationship between TERE1/UBIAD1 and Hras.The results of Drosophila heix gene regulation study indicate that the Drosophila heix gene promoter is located between the upstream 170bp and downstream 350bp of the transcription initiation site. The Drosophila heix gene promoter contains several transcriptional regulatory elements, and has high transcriptional activity. There are specific microRNA target sites in the 3 `UTR region of Drosophila heix gene. Heix gene expression may be post-transcriptional regulated by several Drosophila microRNAs, such as dmiRNA3,dmiRNA309,dmiRNA318,dmiRNA4,dmiRNA133 and dmiRNA79.Our research results have laid a good foundation for the further study of the function and expression regulation of heix gene.更多还原