Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1
Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1
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DOI：
10.1111/j.1471-4159.2004.02447.x
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发表时间：
2004-06-01
影响因子：
4.7
通讯作者：
Haass, C
中科院分区：
文献类型：
--
作者：
Shirotani, K;Edbauer, D;Haass, C
γ-分泌酶是一种高分子质量天冬氨酰蛋白酶复合物，由早老素（PS1 或 PS2）、尼卡斯特林 (Nct)、前咽缺陷-1 (APH-1) 和早老素增强剂-2 (PEN-2) 组成。该复合物介导 β 分泌酶裂解 β 淀粉样前体蛋白 (APP) 的膜内蛋白水解，导致阿尔茨海默病相关淀粉样 β 肽 (Abeta) 的分泌。为了剖析γ-分泌酶复合物组装、成熟和活性所需的Nct的功能重要结构域，我们对进化保守氨基酸进行了突变。突变的 Nct 变体在内源性 Nct 显着减少的细胞背景中表达。通过恢复 PS、APH-1 和 PEN-2 表达的能力以及监测 APP C 末端片段（γ 分泌酶的直接底物）的积累，对突变体 Nct 进行了功能研究。我们在 Nct 胞外域内发现了三个独立的突变，它们挽救了 APH-1 的表达，但不能挽救 PEN-2 或 PS 的表达，因此未能恢复 γ 分泌酶活性。有趣的是，这些不成熟的 Nct 变体选择性地结合 APH-1，表明 Nct/APH-1 存在稳定的相互作用，不依赖于 PS 和 PEN-2。与这一发现一致，在不存在 PS 和 PEN-2 的情况下，PS 双敲除和与 APH-1 共免疫沉淀的未成熟 Nct 中 APH-1 的表达基本不受影响。综上所述，我们的研究结果表明，未成熟的 Nct 可以稳定地与 APH-1 相互作用，形成 PS 和 PEN-2 结合的潜在支架。此外，后两个复杂伙伴的结合关键取决于 Nct 胞外域的完整性。
gamma-Secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.