Division of Neuroscience and Experimental Psychology, Faculty of Biology, Medicine and Health, School of Biological Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK.
Proteomics Facility, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, UK.
J Neurochem. 2019 May;149(3):399-412. doi: 10.1111/jnc.14666. Epub 2019 Feb 14.
The generation of the amyloid-β (Aβ) peptides from the amyloid precursor protein (APP) through sequential proteolysis by β- and γ-secretases is a key pathological event in the initiation and propagation of Alzheimer's disease. Aβ and the transcriptionally active APP intracellular domain are generated preferentially from the APP695 isoform compared to the longer APP751 isoform. As the Aβ and amyloid precursor protein intracellular domain produced from cleavage of APP695 and APP751 are identical we hypothesised that the two isoforms have differences within their interactomes which mediate the differential processing of the two isoforms. To investigate this, we applied a proteomics-based approach to identify differences in the interactomes of the APP695 and APP751 isoforms. Using stable isotope labelling of amino acids in cell culture and quantitative proteomics, we compared the interactomes of APP695 and APP751 expressed in human SH-SY5Y cells. Through this approach, we identified enrichment of proteins involved in mitochondrial function, the nuclear pore and nuclear transport specifically in the APP695 interactome. Further interrogation of the APP interactome and subsequent experimental validation (co-immunoprecipitation and siRNA knockdown) revealed GAP43 as a specific modulator of APP751 proteolysis, altering Aβ generation. Our data indicate that interrogation of the APP interactome can be exploited to identify proteins which influence APP proteolysis and Aβ production in an isoform dependent-manner. Cover Image for this issue: doi: 10.1111/jnc.14504.
β-和γ-分泌酶通过顺序蛋白水解作用将淀粉样前体蛋白（APP）生成淀粉样-β（Aβ）肽，这是阿尔茨海默病发病和进展的关键病理事件。与较长的 APP751 异构体相比，APP695 异构体通过蛋白水解作用优先生成 Aβ和具有转录活性的 APP 细胞内结构域。由于从 APP695 和 APP751 的切割产生的 Aβ和 APP 细胞内结构域是相同的，我们假设两种异构体在其互作组中有差异，这些差异介导了两种异构体的差异加工。为了研究这一点，我们应用了一种基于蛋白质组学的方法来鉴定 APP695 和 APP751 异构体互作组的差异。我们使用细胞培养中的稳定同位素标记氨基酸和定量蛋白质组学，比较了在人 SH-SY5Y 细胞中表达的 APP695 和 APP751 的互作组。通过这种方法，我们鉴定了富含与线粒体功能、核孔和核转运相关的蛋白质，这些蛋白质在 APP695 互作组中特异性富集。对 APP 互作组的进一步分析和随后的实验验证（免疫共沉淀和 siRNA 敲低）揭示了 GAP43 是 APP751 蛋白水解的特定调节剂，改变了 Aβ的生成。我们的数据表明，对 APP 互作组的研究可以用于鉴定以异构体依赖的方式影响 APP 蛋白水解和 Aβ产生的蛋白质。本期的封面图片：doi: 10.1111/jnc.14504.
