Imai So, Cai Tetsuo, Yoshida Chika, Tomita Taisuke, Futai Eugene
Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramakiazaaoba, Aobaku, Sendai, Miyagi, Japan.
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
J Biochem. 2019 Jan 1;165(1):37-46. doi: 10.1093/jb/mvy081.
γ-Secretase generates amyloid beta peptides (Aβ) from amyloid precursor protein through multistep cleavages, such as endoproteolysis (ε-cleavage) and trimming (γ-cleavage). Familial Alzheimer's disease (FAD) mutations within the catalytic subunit protein of presenilin 1 (PS1) decrease γ-cleavage, resulting in the generation of toxic, long Aβs. Reducing long Aβ levels has been proposed as an AD therapeutic strategy. Previously, we identified PS1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. Here, we analysed these PS1 mutations in the presence of NCT, and found that they were constitutively active in yeast. One triple, 13 double, and 5 single mutants enhanced ε-cleavage activity up to 2.7-fold. Furthermore, L241I, F411Y, S438P and F441L mutations modulated trimming activities to produce more short-Aβ in yeast microsomes. When introduced in mouse embryonic fibroblasts, these mutations possessed similar or reduced ε-cleavage activity. However, two mutations, L241I and S438P, modulated trimming activities and changed the conformation of transmembrane domain 1, the substrate recognition site. These mutants had the opposite modulatory effects of FAD mutations and produced more short Aβs and fewer long Aβs. Our results provide insights into the relationship between PS1 conformational changes and γ-secretase activities.
γ-分泌酶通过多步切割，如内蛋白水解（ε-切割）和修剪（γ-切割），从淀粉样前体蛋白生成淀粉样β肽（Aβ）。早老素1（PS1）催化亚基蛋白内的家族性阿尔茨海默病（FAD）突变会降低γ-切割，导致产生有毒的长Aβ。降低长Aβ水平已被提出作为一种阿尔茨海默病治疗策略。此前，我们使用酵母γ-分泌酶测定法鉴定了在没有尼卡斯特林（NCT）的情况下仍具有活性的PS1突变。在此，我们分析了在有NCT存在时的这些PS1突变，发现它们在酵母中具有组成型活性。一个三联体、13个双突变体和5个单突变体将ε-切割活性提高了2.7倍。此外，L241I、F411Y、S438P和F441L突变调节了修剪活性，在酵母微粒体中产生更多短Aβ。当引入小鼠胚胎成纤维细胞时，这些突变具有相似或降低的ε-切割活性。然而，两个突变L241I和S438P调节了修剪活性并改变了跨膜结构域1（底物识别位点）的构象。这些突变体具有与FAD突变相反的调节作用，产生更多短Aβ和更少长Aβ。我们的结果为PS1构象变化与γ-分泌酶活性之间的关系提供了见解。
