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.

许多膜蛋白的细胞外结构域可以蛋白水解裂解，从而导致释放到培养基中。在生长因子和其他膜蛋白中观察到了这种蛋白水解加工，也称为“胞外域脱落”。膜蛋白的外生域脱落会影响膜蛋白的生物学活性。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。可溶性成熟的HB-EGF是由胞外域脱落从proHB-egf产生的。 TPA是HB-EGF外生域脱落的强刺激者，Adam家族金属蛋白酶也参与了这种裂解。然而，膜蛋白的胞外域脱落的机制仍然完全晦涩。为了了解TPA诱导的proHB-EGF胞外域的脱落的机制，我们试图鉴定出一种新的S蛋白质候选者，该蛋白质与HB-EGF外生域通过使用慢性病毒sirna库中的功能遗传筛查有关。用TPA处理后，收集表达强PROHB-EGF信号的细胞，并从细胞中回收RNA，并用PCR用特异性慢病毒载体的寡核苷酸扩增。鉴定出在选择过程中特异性浓缩的siRNA模板。三个独立的筛选表明，有10个以上的基因是参与HB-EGF脱落的候选基因。 ADAM17也包括在这10个基因中。这些结果表明，siRNA文库的功能丧失遗传筛选对于确定目的的基因很有用。