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Proteome Analysis of Disease-related Genes based on Alternative Splicing

基于选择性剪接的疾病相关基因的蛋白质组分析

基本信息

批准号：
16201042
负责人：
YANAGAWA Hiroshi
金额：
$ 30.04万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

项目摘要

(1) Computational analysis of disease-related genes with splice variants: We searched genes containing both ASDB (database of alternatively spliced genes) and OMIM (Online Mendelian Inheritance in Man) databases to extract alternatively spliced genes that related to diseases such as cancer and Alzheimer-disease.(2) Construction of a parallel IVV screening system: We optimized the conditions of IVV screening for protein-protein interactions (PPIs) in 96-well microplates with a QIAGEN BioRobot 8000. We also improved the performance of computer systems for analysis of the huge sequence data (from 1000 sequences per day to 16000 sequences per day).(3) A large-scale analysis of PPIs : As a result of the parallel IVV screening against 68 distinct bait proteins, total of -1000 PPIs were successfully detected. When arbitrary chosen PPIs were tested by pull-down assays, -70% of PPIs were verified.(4) Confirmation of PPIs with splice variants : Among the above PPI data, 24 gene fragments contain their protein-binding regions within variable regions derived from alternative splicing. We confirmed that the in vitro binding ability of these genes was certainly affected by the change of splicing patterns.(5) Construction of a PPI database : We developed a database containing not only the binding region from the PPI data but also the variable region of alternative splicing and SNP, the crystal structure of protein complexes, gene localization, etc. The database is useful for analyzing the effect of alternative splicing on disease-related gene networks.

(1)具有剪接变体的疾病相关基因的计算分析：我们搜索包含ASDB（选择性剪接基因数据库）和OMIM（在线孟德尔遗传人类）数据库的基因，以提取与癌症和阿尔茨海默病等疾病相关的选择性剪接基因。(2)构建一个平行的IVV筛选系统：我们用QIAGEN BioRobot 8000优化了96孔微孔板中蛋白质-蛋白质相互作用（PPI）的IVV筛选条件。我们还提高了计算机系统的性能，用于分析巨大的序列数据（从每天1000个序列到每天16000个序列）。(3)PPI的大规模分析：作为针对68种不同诱饵蛋白的平行IVV筛选的结果，成功检测到总共约1000种PPI。当通过下拉试验检测任意选择的PPI时，约70%的PPI得到验证。(4)PPI与剪接变体的确认：在上述PPI数据中，24个基因片段在可变区中含有其蛋白结合区，可变区来源于选择性剪接。我们证实了这些基因的体外结合能力肯定会受到剪接模式变化的影响。(5)PPI数据库的构建：我们开发了一个数据库，其中不仅包含PPI数据的结合区域，还包含选择性剪接和SNP的可变区、蛋白质复合物的晶体结构、基因定位等。该数据库可用于分析选择性剪接对疾病相关基因网络的影响。