Laboratory of Enzymology, Graduate School of Agricultural Sciences, Tohoku University, Sendai 980-0845, Japan.
Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Int J Mol Sci. 2022 Jan 3;23(1):507. doi: 10.3390/ijms23010507.
Amyloid beta peptides (Aβs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aβ42/43 species is enhanced by familial Alzheimer's disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aβ production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aβs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.
淀粉样β肽（Aβs）通过 γ-分泌酶介导的多种切割步骤从淀粉样前体蛋白（APP）产生，包括内切蛋白酶和羧肽酶样修剪。家族性阿尔茨海默病（FAD）突变使 γ-分泌酶的催化亚基早老素 1（PS1）内的神经毒性 Aβ42/43 物种的产生增强。PS1 的 FAD 突变导致部分功能丧失并降低切割活性。具有 FAD 突变相反效果的激活突变对于研究 Aβ 产生很重要。Aph1 是 γ-分泌酶的调节亚基；它被认为是复合物的支架。在这项研究中，我们使用酵母 γ-分泌酶测定法鉴定了在没有 nicastrin（NCT）的情况下具有活性的 Aph1 突变。我们在存在 NCT 的情况下分析了这些 Aph1 突变，发现 L30F/T164A 突变是激活的。当引入小鼠胚胎成纤维细胞时，该突变增强了切割。与野生型 Aph1 相比，突变体产生了更多的短和长 Aβs，而没有明显的调节功能。突变体没有改变 γ-分泌酶复合物的数量，这表明 L30F/T164A 增强了催化活性。我们的结果提供了 Aph1 在 γ-分泌酶活性中的调节功能的见解。
