In vivo reconstitution of gamma-secretase in Drosophila results in substrate specificity.
In vivo reconstitution of gamma-secretase in Drosophila results in substrate specificity.
复制标题
果蝇中γ-分泌酶的体内重建导致底物特异性。
DOI：
10.1128/mcb.00030-10
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发表时间：
2010
影响因子：
5.3
通讯作者：
Merdes,Gunter
中科院分区：
文献类型：
--
作者：
Stempfle,Denise;Kanwar,Ritu;Loewer,Alexander;Fortini,MarkE;Merdes,Gunter
膜内天冬氨酸蛋白酶γ-分泌酶在参与细胞分化的几个信号通路中起着重要作用，并与包括阿尔茨海默病在内的多种人类疾病有关。在这里，我们描述了体内重组γ分泌酶的转基因模型，该模型基于四个核心γ分泌酶组分：早老素、尼卡斯特素、APH-1和PEN-2的表位标记版本的表达。与之前的细胞培养和酵母研究一致，这四种成分的共同表达促进了成熟的、具有蛋白分解活性的γ分泌酶的有效组装。我们证明了体内重组的γ分泌酶具有生化特性和类似于内源性γ分泌酶的亚细胞分布。然而，在转基因苍蝇实验中对替代底物的切割分析表明，重组γ分泌酶对不同底物的活性存在意想不到的功能差异，包括与Notch受体的切割相比，一些APP家族成员的切割明显减少。这些发现表明，在生活的生理条件下，附加因素对其底物的γ分泌酶活性有不同的调节作用。因此，我们的方法首次证明了重组γ分泌酶在多细胞生物体中的整体功能，以及在某些底物的活体切割中对底物特定因子的要求。
The intramembrane aspartyl protease γ-secretase plays a fundamental role in several signaling pathways involved in cellular differentiation and has been linked with a variety of human diseases, including Alzheimer's disease. Here, we describe a transgenicDrosophilamodel forin vivo-reconstituted γ-secretase, based on expression of epitope-tagged versions of the four core γ-secretase components, Presenilin, Nicastrin, Aph-1, and Pen-2. In agreement with previous cell culture and yeast studies, coexpression of these four components promotes the efficient assembly of mature, proteolytically active γ-secretase. We demonstrate thatin vivo-reconstituted γ-secretase has biochemical properties and a subcellular distribution resembling those of endogenous γ-secretase. However, analysis of the cleavage of alternative substrates in transgenic-fly assays revealed unexpected functional differences in the activity of reconstituted γ-secretase toward different substrates, including markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor. These findings indicate thatin vivounder physiological conditions, additional factors differentially modulate the activity of γ-secretase toward its substrates. Thus, our approach for the first time demonstrates the overall functionality of reconstituted γ-secretase in a multicellular organism and the requirement for substrate-specific factors for efficientin vivocleavage of certain substrates.