Post-genome analysis on proteolytic systems of multicellular organism

多细胞生物蛋白水解系统的基因组后分析

• 批准号: 13680725
• 负责人: INOUE Hideshi
• 金额: $ 2.3万
• 依托单位: Tokyo University of Pharmacy and Life Science Grant-in Aid for Scientific Research ( C ) (2)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680725/
• 关键词: C. elegans; protease; RNAi; proteolysis; RNAi; プロテアーゼインビター; ポストゲノム; プロテアソーム; ADAMTS

C. elegans has over 300 genes that code for protease-like proteins. We carried out RNAi analysis on about 190 protease-like genes. Proteases essential for the life cycle of C. elegans were found to be as follows: proteasome, signal peptidase, Lon protease, a cathepsin L-like cysteine protease, a trypsin-like serine protease, some of the ubiquitin-specific proteases, mitochondria processing protease and so on. RNAi on these genes led to lethality. Most of the proteasome subunits except for Rpn9, Rpn10, and Rpn12 are essential. Simultaneous interference of Rpn10 and Rpn12 caused lethal effect. Additionally, we found a novel ADAMTS protease essential for morphogenesis. Two metalloproteases that have a CUB domain and a TSP-1 domain were required for molting. Six serine proteases homologous to dipeptidyl peptidase IV or acylaminoacyl peptidase were found to participate in migration of distal tip cells of gonad. In order to reveal the distribution of these proteases, we performed GFP-expression analysis, in situ hybridization, immunostaining, etc. Some of the proteases were enzymologically characterized using proteins prepared by heterologous expression system or purified from C. elegans

秀丽隐杆线虫具有300多种基因，可为蛋白酶样蛋白编码。我们对大约190种蛋白酶样基因进行了RNAi分析。发现秀丽隐杆线虫生命周期必不可少的蛋白酶如下：蛋白酶体，信号肽酶，LON蛋白酶，组织蛋白酶L样的半胱氨酸蛋白酶，一种胰蛋白酶样丝氨酸蛋白酶，一些泛素蛋白特异性蛋白酶，线粒体加工蛋白酶的某些蛋白酶。这些基因上的RNAi导致了致命性。除RPN9，RPN10和RPN12外，大多数蛋白酶体亚基都是必不可少的。 RPN10和RPN12的同时干扰会导致致命作用。此外，我们发现了一种新型的ADAMTS蛋白酶，对形态发生必不可少。摩尔需要两个具有CUB结构域和TSP-1结构域的金属蛋白酶。发现六个丝氨酸蛋白酶同源于二肽基肽酶IV或酰基氨基氨基酰基肽酶参与性腺远端细胞的迁移。为了揭示这些蛋白酶的分布，我们进行了GFP表达分析，原位杂交，免疫染色。