Karolinska Institutet Dainippon Sumitomo Pharma Alzheimer Center, KI Alzheimer Disease Research Center, Department of Neurobiology, Karolinska Institutet, Novum, Huddinge SE-141 57, Sweden.
J Biol Chem. 2012 Apr 6;287(15):11991-2005. doi: 10.1074/jbc.M111.246074. Epub 2012 Feb 7.
In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.
在阿尔茨海默病中，寡聚淀粉样β肽（Aβ）导致突触丧失和神经元死亡。γ-分泌酶是一种跨膜蛋白酶复合物，介导将 Aβ 从其前体蛋白（APP）中释放出来的最后一步催化反应，它有许多底物，旨在减少 Aβ 产生的治疗剂理想情况下应该针对 APP 切割具有特异性。已经表明 APP 可以在脂筏中进行加工，并且 γ-分泌酶相关蛋白可以影响 Aβ 的产生。在这里，我们使用生物素化抑制剂进行亲和纯化 γ-分泌酶和相关蛋白，并使用质谱法鉴定纯化的蛋白，我们在脑的去污剂抗性膜中鉴定了新的 γ-分泌酶相关蛋白。此外，我们通过小干扰 RNA 介导的基因表达敲低表明，γ-分泌酶相关蛋白的一部分，特别是电压依赖性阴离子通道 1（VDAC1）和接触蛋白相关蛋白 1（CNTNAP1），可使 Aβ 的产生（Aβ40 和 Aβ42）减少约 70%，而 γ-分泌酶复合物的必需组成部分之一的早老素 1 的敲低可使 Aβ 的产生减少 50%。重要的是，这些蛋白对 Notch 加工的影响较小。我们得出结论，VDAC1 和 CNTNAP1 在去污剂抗性膜中与 γ-分泌酶结合，并影响 APP 的加工，并表明干扰这种相互作用的分子可能对阿尔茨海默病具有治疗用途。
