Identification of genes involved in the ectodomain sheddingof HB-EGF

HB-EGF胞外域脱落相关基因的鉴定

基本信息

批准号：
18370079
负责人：
MEKADA Eisuke
金额：
$ 11.05万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18370079/
关键词：
ectodomain shedding SiRNA HB-EGF DNA aray シェディングレンチウィルス

项目摘要

The extracellular domain of a number of membrane proteins can be proteolytically cleaved causing release into the medium. This proteolytic processing, also referred to as "ectodomain shedding", is observed in growth factors and other membrane proteins. Ectodomain shedding of membrane proteins affects the biological activities of membrane proteins. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family. The soluble mature HB-EGF is generated from proHB-EGF by ectodomain shedding. TPA is a strong stimulater of HB-EGF ectodomain shedding, and ADAM family metalloprotease is also involved in this cleavage. However, the mechanism of the ectodomain shedding of membrane proteins remains totally obscure. To understand the mechanism of the TPA-induced shedding of the proHB-EGF ectodomain, we attempted to identify a new candidate of s proteins which is involved in the HB-EGF ectodomain shedding by loss-of-function genetic screenings using Lentiviral siRNA Library. After treatment with TPA, cells expressing strong proHB-EGF signal were collected, and RNA is recovered from the cells and amplified by PCR with oligonucleotides specific to the lentiviral vector. siRNA templetes which were specifically concentrated during selection were identified. Three independent screenings suggested that more than 10 genes are candidate genes involved in HB-EGF shedding. ADAM17 was also included in these 10 genes. These results indicate that the loss-of-function genetic screening by siRNA library is useful to identify genes for the purpose.

许多膜蛋白的胞外结构域可以被蛋白水解切割，导致释放到培养基中。这种蛋白水解过程也称为“胞外域脱落”，在生长因子和其他膜蛋白中观察到。膜蛋白的胞外域脱落影响膜蛋白的生物活性。肝素结合 EGF 样生长因子 (HB-EGF) 是表皮生长因子 (EGF) 家族的成员。可溶性成熟 HB-EGF 是由 proHB-EGF 通过胞外域脱落产生的。 TPA 是 HB-EGF 胞外域脱落的强烈刺激剂，ADAM 家族金属蛋白酶也参与这种裂解。然而，膜蛋白胞外域脱落的机制仍然完全不清楚。为了了解 TPA 诱导 proHB-EGF 胞外域脱落的机制，我们尝试使用慢病毒 siRNA 文库通过功能丧失遗传筛选来鉴定参与 HB-EGF 胞外域脱落的新候选 s 蛋白。用 TPA 处理后，收集表达强 proHB-EGF 信号的细胞，从细胞中回收 RNA，并使用慢病毒载体特异的寡核苷酸通过 PCR 进行扩增。鉴定了在选择过程中特别浓缩的siRNA模板。三个独立筛选表明超过 10 个基因是参与 HB-EGF 脱落的候选基因。 ADAM17也包含在这10个基因中。这些结果表明，通过 siRNA 文库进行的功能丧失遗传筛选对于鉴定用于此目的的基因是有用的。