NOVEL APPROACH FOR EXPRESSION CLONING OF APP SECRETASES

应用秘密表达克隆的新方法

• 批准号: 2408484
• 负责人: KUMAR SAMBAMURTI
• 金额: $ 7.05万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-15 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2408484
• 关键词: HeLa cells; Schizosaccharomyces pombe; alkaline phosphatase; amyloid proteins; cell transformation; chemical cleavage; complementary DNA; endopeptidases; enzyme activity; fungal genetics; gene complementation; gene expression; gene targeting; molecular cloning; nucleic acid sequence; plasmids; polymerase chain reaction

The amyloid beta protein is an approximately 4 kD secreted protein normally found in human plasma and cerebrospinal fluid. Abeta is invariably deposited as insoluble amyloid fibrils in the brains of patients with Alzheimer's disease (AD), and there is increasing evidence that Abeta deposition plays an important role in AD pathogenesis. Abeta is released from the larger amyloid beta protein precursor (betaAPP) through cleavage on the amino and carboxyl side of Abeta by proteolytic activities referred to as beta and gamma secretase, respectively. betaAPP is also cleaved at Abeta 16 by a third secretase, a secretase, which may serve to prevent amyloid deposition by bisecting the Abeta peptide. Despite their important role in Alzheimer's disease, none of the betaAPP secretases has yet been isolated or cloned. Here, I propose to isolate betaAPP secretase cDNAs using a system in which the fission yeast Schizosaccharomyces pombe expresses a chimeric cDNA construct consisting of a secreted derivative of the human placental alkaline phosphatase (SEAP) linked to the C-terminal 105 amino acids of betaAPP (SEAP-APP). I have shown that the secretases in mammalian cells readily process SEAP-APP so that Abeta and secreted SEAP-containing derivatives (sSEAP-APP) are efficiently secreted. In contrast, S. pombe expressing SEAP-APP does not produce secreted derivatives indicating that it lacks endogenous secretase activity. In my initial studies, S. pombe cells expressing SEAP-APP were transformed with a human cDNA (HeLa cell) expression library, and 80,000 transformants, each of which could contain several cDNAs, were screened for their ability to produce secreted derivatives. Five transformants contained putative secretase cDNAs that resulted in the production of sSEAP-APP. Thus complementation in S. pombe appears to be an effective and convenient method to isolate putative human secretase cDNAs. In this application, I propose to employ this system and modifications of it to isolate as many independent candidate betaAPP-secretase cDNAs as possible. I will then determine which of these cDNAs encode authentic betaAPP secretases by determining whether coexpression with authentic betaAPP in S. pombe results in cleavage at the predicted site(s).

淀粉样β蛋白是一种约4kD的分泌蛋白 通常存在于人血浆和脑脊液中。 Abeta是 总是以不溶性淀粉样纤维的形式沉积在大脑中 阿尔茨海默病（AD）患者，并且有越来越多的证据表明 A β沉积在AD发病机制中起重要作用。Abeta β-淀粉样蛋白前体（betaAPP） 通过蛋白水解在A β的氨基和羧基侧上裂解， 分别称为β和γ分泌酶的活性。 β APP还被第三种分泌酶，分泌酶， 其可通过将A β一分为二来防止淀粉样蛋白沉积， 肽。尽管它们在阿尔茨海默病中起着重要作用， β APP分泌酶还没有被分离或克隆。在这里，我建议 为了分离β APP分泌酶cDNA，使用了一种系统， 酵母裂殖酵母表达嵌合cDNA构建体 由人胎盘碱性蛋白的分泌衍生物组成 与betaAPP的C-末端105个氨基酸连接的磷酸酶（SEAP） （SEAP-APP）。我已经证明哺乳动物细胞中的分泌酶 加工SEAP-APP，使得Abeta和分泌的含SEAP的衍生物 （sSEAP-APP）被有效地分泌。相反，S.粟酒表达 SEAP-APP不产生分泌衍生物，表明其缺乏 内源性分泌酶活性。在我最初的研究中，S。粟球细胞 用人cDNA转化表达SEAP-APP的细胞（HeLa细胞） 表达文库和80，000个转化体，每个转化体都可以 包含几个cDNA，筛选它们的能力， 分泌的衍生物。5个转化体含有推定的分泌酶 因此，互补的cDNA导致sSEAP-APP的产生。 in S.粟酒裂殖酵母似乎是一种有效和方便的方法， 推测的人类分泌酶cDNA。在本申请中，我建议雇用 这个系统和它的修改隔离尽可能多的独立 候选β APP-分泌酶cDNA。然后我会决定 通过确定这些cDNA中哪一个编码真正的β APP分泌酶， 是否与S.粟酒导致 在预测位点处的切割。

项目成果

Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

糖基磷脂酰肌醇锚定蛋白在阿尔茨海默病淀粉样β蛋白的生物发生中发挥重要作用。

• DOI: 10.1074/jbc.274.38.26810
• 发表时间: 1999
• 期刊: The Journal of biological chemistry
• 作者: Sambamurti,K;Sevlever,D;Koothan,T;Refolo,LM;Pinnix,I;Gandhi,S;Onstead,L;Younkin,L;Prada,CM;Yager,D;Ohyagi,Y;Eckman,CB;Rosenberry,TL;Younkin,SG
• 通讯作者: Younkin,SG