Beta-secretase inhibitor potency is decreased by aberrant beta-cleavage location of the “Swedish mutant” amyloid precursor protein
Beta-secretase inhibitor potency is decreased by aberrant beta-cleavage location of the “Swedish mutant” amyloid precursor protein
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DOI：
10.1016/j.jalz.2010.05.1791
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发表时间：
2010-07
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影响因子：
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通讯作者：
Hidekuni Yamakawa;Sosuke Yagishita;E. Futai;S. Ishiura
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作者：
Hidekuni Yamakawa;Sosuke Yagishita;E. Futai;S. Ishiura
淀粉样蛋白-β (Aβ)肽被广泛认为是阿尔茨海默病（AD）的致病分子，是由天门氨酸蛋白酶BACE1/β-分泌酶和早老素/γ-分泌酶连续切割淀粉样蛋白前体蛋白（APP）产生的。因此，抑制BACE1是一种很有希望的预防AD进展的策略。然而，β-分泌酶抑制剂（bsi）在表达“瑞典突变体”APP （APPswe）的细胞和过表达APPswe的转基因小鼠Tg2576中表现出出乎意料的低效力。瑞典突变显著加速了APP的β-裂解，从而产生了Aβ；这种加速被认为是BSI对APPswe加工的抑制活性较差的原因，但详细的机制仍有待阐明。方法采用酶法和细胞法研究了BSI在不同条件下对BACE1处理瑞典型和野生型底物的影响。令人惊讶的是，与基于细胞的实验相比，使用纯化的BACE1和底物进行的酶促实验没有观察到BSI效力的降低，这表明瑞典突变导致的β-裂解加速不是其根本原因。在进行了几次实验后，我们发现，通过在c端引入Y687A突变，当细胞区室被破坏，APP的β -裂解位置被限制在秘书囊泡中时，基于细胞的检测并未观察到效力下降。结论sbsi效力的降低是由于APPswe加工的异常亚细胞定位，而不是由于β-裂解的加速或β-裂解的APP c端片段的积累。由于大多数散发性AD患者表达野生型APP，我们的研究结果表明野生型小鼠优于Tg2576小鼠作为确定AD患者BSI有效剂量的模型。本研究为BSI的效价降低提供了新的见解，并为其作为一种疾病调节剂的发展提供了有价值的建议。
BackgroundThe amyloid-β (Aβ) peptide, widely known as the causative molecule of Alzheimer's disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/β-secretase and presenilin/γ-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, β-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing “Swedish mutant” APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates β-cleavage of APP and hence the generation of Aβ; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing, but the detailed mechanism remains to be elucidated.MethodsWe examined the efficacies of BSI on the processing of Swedish and wild type substrates by BACE1 under several conditions in the enzymatic and cell-based assays.ResultsSurprisingly, decreased BSI potency was not observed in an enzymatic assay using purified BACE1 and substrates in contrast to a cell-based assay, indicating that the accelerated β-cleavage resulting from the Swedish mutation is not its underlying cause. Following several experiments focusing on differences between the enzymatic and cell-based assays, we found that the potency decrease in a cell-based assay was not observed when the cellular compartment was disrupted and when the location of beta-cleavage of APP was limited in secretary vesicles by introducing the Y687A mutation into the C-terminus.ConclusionsBSI potency is decreased by the aberrant subcellular localization of APPswe processing and not by accelerated β-cleavage or the accumulation of the C-terminal fragment of β-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.