Keller Preston C, Tomita Taisuke, Hayashi Ikuo, Chandu Dilip, Weber Jason D, Cistola David P, Kopan Raphael
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8231, St. Louis, Missouri 63110, USA.
Biochemistry. 2006 Apr 25;45(16):5351-8. doi: 10.1021/bi052228a.
Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for gamma-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized gamma-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without gamma-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the gamma-secretase specific cleavage products. In addition, we show that addition of aspartyl- and gamma-secretase specific protease inhibitors are able to retard the formation of these variants independent of gamma-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized gamma-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.
膜内蛋白水解是一个新兴且快速发展的领域。利用重组底物对γ-分泌酶（一种膜内切割酶）进行体外测定，对于表征这种特殊酶的生化特性至关重要。几种不同长度的重组Notch蛋白通常用作CHAPSO溶解的γ-分泌酶的体外底物。在此我们报告，几种重组Notch构建体在体外经历有限的蛋白水解或根本不发生蛋白水解。相反，在与γ-分泌酶一起孵育或不孵育的情况下，完整蛋白的变体在SDS-PAGE期间迁移到γ-分泌酶特异性切割产物预期的位置。此外，我们表明添加天冬氨酰和γ-分泌酶特异性蛋白酶抑制剂能够独立于γ-分泌酶延迟这些变体的形成，这可能导致错误的结论，即即使没有发生蛋白水解，溶解的γ-分泌酶对Notch的切割也在体外实现了。相比之下，在哺乳动物或昆虫细胞中产生的底物在体外能被有效切割。这些观察结果表明，依赖重组细菌产生的Notch跨膜结构域的体外研究应在包含能够区分实际切割和这种潜在假象的额外对照的情况下进行。
