兰州大学机构库 >生命科学学院
对PRMT5几个缺失突变体的功能及其与GM130之间相互作用的研究
Alternative TitleStudy on biological function of various truncations of human PRMT5 in E.coli and direct interaction with GM130
孙力涛
Thesis Advisor谢小冬
2008-05-24
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordPRMT5 GM130 纯化 甲基转移酶活性
Abstract蛋白精氨酸甲基化是一种普遍存在于细胞核和细胞质中的翻译后修饰现象,由精氨酸甲基转移酶家族(PRMTs)催化产生。越来越多的实验证明蛋白甲基化参与了生命活动的各个过程,包括RNA的加工和转运、蛋白翻译、基因转录、信号转导和DNA修复等。大多数PRMTs作用于底物中的赖氨酸和精氨酸的富集区(GAR motifs)。根据将S-腺苷甲硫氨酸(S-adenosylmethionine,AdoMet)上的甲基转移到精氨酸的胍基氮上产生甲基化的不同方式,最终可以分为三类:单甲基化(MMA)、对称性双甲基化(sDMA)和非对称性双甲基化(aDMA)。 PRMTs可以分为类型I、II、III和IV四种类型。I类和II类都可以催化产生单甲基化(MMA),不同点在于:I类(PRMT1, 3, 4, 6和8)催化产生非对称性双甲基化(aDMA),而II类(PRMT5, PRMT7和FBXO11)催化产生对称性双甲基化(sDMA),PRMT7也呈现III类酶的特点,在一些底物倾向于催化产生单甲基化(MMA),但是并不产生对称性双甲基化(sDMA),IV型酶在酵母中催化瓜基上的单原子氮。 蛋白质精氨酸甲基转移酶5 (PRMT5)在细胞生长和信号转导方面是一个重要的调节因子,主要参与染色质重塑、RNA剪切、基因转录、细胞分化等过程。因此,对其结构和功能的研究就显得十分重要。通过大肠杆菌表达系统把全长基因PRMT5构建到pGEX-4T-1表达载体上,所得到GST标签的重组蛋白可溶性很低。为此,通过在其N端缺失不同氨基酸序列来增加其表达量,而且其中有一个缺失突变体的活性并没有发生改变。同时,我们还发现PRMT5的N端的前15个氨基酸对其甲基转移酶的催化活性很重要。 高尔基体在蛋白翻译后修饰,蛋白分类和运输发挥着重要作用,而在维持高尔基体的结构和控制高尔基体组装方面,依赖于两类重要的蛋白,即golgin蛋白和GRASPs (Golgi Re-Assembly Stacking Proteins)蛋白。其中GRASPs也称为Golgi matrix蛋白,包括Giantin, GM130, GRASP6,它们都是维持高尔基的组装所必须的蛋白。蛋白的翻译后修饰,例如GM130的磷酸化对高尔基体的结构十分重要。本实验室已经发现在Hela细胞质中分离提取了PRMT5复合体,并对其中的几个重要组分进行了质谱分析,其中一个在高尔基体结构和功能中发挥重要作用的组分GM130。为了证明PRMT5和GM130之间的相互作用,我们进行了Farwestern实验。实验结果显示,GM130 通过其N端的前73个氨基酸结合于PRMT5的保守区域(C端)。
Other AbstractArginine methylation is a prevalent post-translational modification found on both nuclear and cytoplasmic proteins.The methylation of arginine residues is catalyzed by the protein arginine Nmethyltransferase(PRMT) family of enzymes. Previously studies showed Arginine methylation participated in pre-mRNA processing, protein translation ,DNA transcription, signal transduction and DNA repair. Most PRMTs methylate glycine- and arginine-rich patches (GAR motifs) within their substrates. The complexity of the methylarginine mark is enhanced by the ability of this residue to be methylated in three different ways on the guanidino group: monomethylated (MMA), symmetrically dimethylated (sDMA) and asymmetrically dimethylated (aDMA), each of which has potentially different functional consequences. PRMTs are classified as type I, type II, type III or type IV enzymes. Type I and type II enzymes all catalyze the formation of an MMA intermediate, then type I PRMTs (PRMT1, 3, 4, 6 and 8)further catalyze the production of aDMA, whereas type II PRMTs (PRMT5, PRMT7 and FBXO11)catalyze the formation of sDMA. PRMT7 also exhibits type III enzymatic activity – the propensity to catalyze the formation of MMA on certain substrates and not proceed with sDMA catalysis. A type IV enzyme that catalyzes the monomethylation of the internal guanidino nitrogen atom has been described in yeast. Protein arginine methyltransferase 5 (PRMT5) has been implicated as an important regulator of many cellular processes and signaling pathways, including chromatin remodeling, RNA splicing, DNA transcription, and cell proliferation. Therefore, structural and functional studies on PRMT5 are quite important. The full length of PRMT5 gene was cloned into vector pGEX-4T-1, resulting in only low expression levels in Escherichia coli (E. coli). Here, we showed that the several N-terminal amino acids deletions could result in a significant increase in the amount of soluble fraction, while one of them did not affect the protein-arginine methyltransferase activity. And we also found that the N-terminal 15 amino acids region of PRMT5 may be important for the catalytic activity. The Golgi apparatus (GA) plays a central role in the post-translational modification, sorting, and transportation of proteins. Maintenance of GA structure and function depends on Golgin proteins and GRASPs (Golgi Re-Assembly Stacking Proteins).The posttranslational modification of golgins, such as phosphorylation of GM130, is cri...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221525
Collection生命科学学院
Recommended Citation
GB/T 7714
孙力涛. 对PRMT5几个缺失突变体的功能及其与GM130之间相互作用的研究[D]. 兰州. 兰州大学,2008.
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