A sensitive and quantitative assay for measuring cleavage of presenilin substrates
A sensitive and quantitative assay for measuring cleavage of presenilin substrates
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DOI：
10.1074/jbc.c100649200
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发表时间：
2002-03-01
影响因子：
4.8
通讯作者：
Lundkvist, J
中科院分区：
文献类型：
--
作者：
Karlström, H;Bergman, A;Lundkvist, J
早老素(PS)蛋白是伽马分泌酶活性的组成部分，在阿尔茨海默病的发病机制中起核心作用。在这里，我们提出了一种新的基于细胞的报告基因分析方法，用于量化PS控制的阿尔茨海默病淀粉样前体蛋白(APP)的伽马分泌酶裂解。我们发现，这种方法有几个优点，包括提高了敏感性和特异性，改进了切割的量化，并同时检测了APP中所有的伽玛-分泌酶切割。此外，APP分析可以与记录Notch受体伽马分泌酶裂解的类似分析平行使用。利用这些分析方法分析了两种已知的伽玛分泌酶抑制剂和假定的PS活性位点突变体对APP和Notch加工的影响，表明可以快速识别不同影响Notch和APP切割的抑制剂和突变体。利用这些检测方法同时高通量筛选APP和Notch的候选伽马分泌酶抑制剂的可能性，为系统地寻找选择性阻断APP切割而不影响Notch信号转导的新型抑制剂开辟了新的前景。
The presenilin (PS) proteins are components of the gamma-secretase activity, which is central in the pathogenesis of Alzheimer's disease. Here we present a novel cell-based reporter gene assay for the quantification of PS-controlled gamma-secretase cleavage of the Alzheimer amyloid precursor protein (APP). We show that this assay offers several advantages, including increased sensitivity and specificity, improved quantification of cleavage, and simultaneous detection of all gamma-secretase cleavages in APP. Furthermore, the APP assay can be used in parallel with a similar assay that records gamma-secretase cleavage of a Notch receptor. The use of these assays to analyze the effects of two known gamma-secretase inhibitors and postulated PS active site mutants on APP and Notch processing demonstrated that inhibitors and mutants that differently affect Notch and APP cleavage can be identified rapidly. The possibility in using these assays for high throughput screening of candidate gamma-secretase inhibitors for APP and Notch in parallel opens up new vistas to systematically search for novel inhibitors that selectively block APP cleavage while not affecting Notch signaling.