Functional Proteomics, Medizinisches Proteom-Center, Ruhr-University Bochum, D-44801 Bochum, Germany.
Mol Cell Proteomics. 2012 Nov;11(11):1274-88. doi: 10.1074/mcp.M112.019364. Epub 2012 Aug 9.
Central hallmark of Alzheimer's disease are senile plaques mainly composed of β-amyloid, which is a cleavage product of the amyloid precursor protein (APP). The physiological function of APP and its family members APLP1 and APLP2 is poorly understood. In order to fill this gap, we established a cell-culture based model with simultaneous knockdown of all members of the family. A comprehensive proteome study of the APP/APLP1/APLP2 knockdown cell lysates versus controls revealed significant protein abundance changes of more than 30 proteins. Targeted validation of selected candidates by immunoblotting supported the significant down-regulation of the methionine adenosyltransferase II, alpha (MAT2A) as well as of peroxiredoxin 4 in the knockdown cells. Moreover, MAT2A was significantly down-regulated at the mRNA level as well. MAT2A catalyzes the production of S-adenosylmethionine from methionine and ATP, which plays a pivotal role in the methylation of neurotransmitters, DNA, proteins, and lipids. MAT2A-dependent significant up-regulation of S-adenosylmethionine was also detectable in the knockdown cells compared with controls. Our results point to a role of the APP family proteins in cellular methylation mechanisms and fit to findings of disturbed S-adenosylmethionine levels in tissue and CSF of Alzheimer disease patients versus controls. Importantly, methylation plays a central role for neurotransmitter generation like acetylcholine pointing to a crucial relevance of our findings for Alzheimer's disease. In addition, we identified differential gene expression of BACE1 and PSEN1 in the knockdown cells, which is possibly a consequence of MAT2A deregulation and may indicate a self regulatory mechanism.
阿尔茨海默病的中心标志是主要由β-淀粉样蛋白组成的老年斑,β-淀粉样蛋白是淀粉样前体蛋白(APP)的裂解产物。APP 及其家族成员 APLP1 和 APLP2 的生理功能知之甚少。为了填补这一空白,我们建立了一种基于细胞培养的模型,同时敲低了该家族的所有成员。对 APP/APLP1/APLP2 敲低细胞裂解物与对照的全面蛋白质组学研究表明,超过 30 种蛋白质的丰度发生了显著变化。通过免疫印迹对选定候选物进行的靶向验证支持了在敲低细胞中甲硫氨酸腺苷转移酶 II,α(MAT2A)以及过氧化物还原酶 4 的显著下调。此外,MAT2A 的 mRNA 水平也显著下调。MAT2A 催化蛋氨酸和 ATP 生成 S-腺苷甲硫氨酸,在神经递质、DNA、蛋白质和脂质的甲基化中起关键作用。与对照相比,敲低细胞中也可检测到 MAT2A 依赖性 S-腺苷甲硫氨酸的显著上调。我们的结果表明 APP 家族蛋白在细胞甲基化机制中起作用,并与阿尔茨海默病患者组织和 CSF 中 S-腺苷甲硫氨酸水平紊乱的发现相吻合。重要的是,甲基化在乙酰胆碱等神经递质的产生中起核心作用,这表明我们的发现对阿尔茨海默病具有至关重要的意义。此外,我们还在敲低细胞中鉴定到 BACE1 和 PSEN1 的差异基因表达,这可能是 MAT2A 失调的结果,可能表明存在一种自我调节机制。