权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analyses of Physiological Functions of Protein-Modifying Enzyme Trabsglutaminase

蛋白修饰酶反谷氨酰胺酶的生理功能分析

基本信息

批准号：
15580077
负责人：
IKURA Koji
金额：
$ 2.5万
依托单位：
Kyoto Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

In order to elucidate physiological functions of protein-modifying enzyme transglutaminase at the molecular level, analyses of its protein substrates and interacting proteins were done, and ectopic gene expressing experiments using Drosophila as a model system were designed.1.It was conmirmed that betaine-homocysteine S-methyltransferase(BHMT) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutaminase. Primary amines were incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. Glutamine residues of BHMT which are reactive for transglutaminase reaction were located in the region near the carboxyl terminal of BHMT subunit, BHMT activity was regulated repressively by the transglutaminase modification, specially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.2.The experiments using immuno-affinity gel beads with monoclonal antibody to guinea pig liver transglutaminase suggested that a 55 kDa protein is a candidate of an interacting protein of tissue-type transglutaminase. Yeast two-hybrid experiments using Sos/Ras-relay system were done to detect proteins interacting with tissue-type transglutaminase in mouse liver. The results suggested that various proteins including enzymes and proteinase inhibitor can be candidates of interacting proteins. We also prepared materials to apply the two-hybrid experiments on human brain.3.A search on the data-base of Drosophila gene (Fly Base) indicated that Drosophila has one transglutaminase gene and two splicing variants are transcribed from the gene. To make the GAL4-UAS expression system of transglutaminase in Drosophila, we are now subcloning the cDNA of Drosophila transglutaminase into pUASP vector.

为了在分子水平上阐明蛋白修饰酶转氨酶的生理功能，对其蛋白底物和相互作用蛋白进行了分析，1.甜菜碱-同型半胱氨酸S-甲基转移酶（betaine-homocysteine S-methyltransferase，BHMT）可以作为组织表达的底物，用猪肝BHMT和豚鼠肝转氨酶进行体外实验。通过转氨酶将伯胺掺入BHMT中。在没有伯胺的情况下，BHMT亚基在分子内和分子间交联。BHMT中与转氨酶反应的谷氨酰胺残基位于BHMT亚基的羧基端附近，BHMT活性受转氨酶修饰的抑制性调节，特别是通过交联作用。该调节反应可能参与了肝脏中同型半胱氨酸代谢的调节。2.用免疫亲和凝胶珠结合抗豚鼠肝转氨酶单克隆抗体的实验表明，一个分子量为55 kDa的蛋白是组织型转氨酶相互作用蛋白的候选者。利用酵母双杂交技术，利用Sos/Ras-relay系统检测小鼠肝脏中与组织型转氨酶相互作用的蛋白。结果表明，包括酶和蛋白酶抑制剂在内的各种蛋白质都可以作为相互作用蛋白的候选者。3.通过对果蝇基因数据库（Fly Base）的检索，发现果蝇有一个转氨酶基因，并从该基因转录出两个剪接变体。为了构建转谷氨酰胺酶的GAL 4-UAS表达系统，我们将果蝇转谷氨酰胺酶的cDNA亚克隆到pUASP载体中。