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Amyloid β-protein production and metabolism of intramembrane regin of APPthrough stepwise processing.

β淀粉样蛋白的产生和APP膜内区域的代谢通过逐步加工。

基本信息

批准号：
18500277
负责人：
MORISHIMA Maho
金额：
$ 2.53万
依托单位：
Hokkaido University (2007)The University of Tokyo (2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

To learn about the molecular mechanism of intramemebrane cleavage of APP by γ-secretase, we have tested the tripeptide hypothesis that Aβ40/42 is produced from longer Aβ that is generated by ε-cleavage through stepwise processing at every three residues along the α-helical structure of the intramembrane region (J Neurosci 25, 436-445, 2005).1. We examined whether longer Aβ (Aβ46) is processed to Aβ43 and Aβ40 in a γ-secretase-dependent manner. CHO cells treated with DAPT caused an accumulation of Aβ46, which was fractionated into lipid raft by sucrose density gradient centrifugation. Incubating the Aβ46-accumulating raft in the presence of L685,458 prevented AICD generation and resulted in a large decrease in the level of Aβ46 and the concomitant appearance of Aβ40 and Aβ43, but not Aβ42. The amount of newly produced Aβ40 and Aβ43 was roughly equivalent to the decrease in the amount of Aβ46. Further addition of DAPT suppressed the production of Aβ40/43 and abolished the decrease in the amount of Aβ46. These data indicate that preaccumulated Aβ46 is processed by γ-secretase to Aβ40/43, but not to Aβ42 in the raft.2. We sought to identify by LC-MS/MS particular tripeptides that should be released from the transmembrane domain of APP according to the tripeptide hypothesis. Using cell -free Aβ generation system with the membrane prepared from CHO cells, we found that the five tripeptides, IAT, VIV, ITL, TVI, and VIT, were generated during incubation. The production of these tripeptides was significantly suppressed by either L685, 458 or DAPT. These results strongly suggest that those five tripeptides are generated by γ-secretase.

为了了解γ-分泌酶对APP的膜内切割的分子机制，我们测试了三肽假说，即Aβ40/42是由较长的Aβ产生的，所述较长的Aβ是通过沿着膜内区域的α-螺旋结构在每沿着三个残基处逐步加工而通过ε-切割产生的（J Neurosci 25，436-445，2005）。我们研究了较长的Aβ（Aβ46）是否以γ-分泌酶依赖性方式加工为Aβ43和Aβ40。经DAPT处理的CHO细胞引起Aβ46蓄积，通过蔗糖密度梯度离心将Aβ46分离成脂筏。在存在L 685，458的情况下孵育Aβ46蓄积筏可防止AICD生成，并导致Aβ46水平大幅降低，同时出现Aβ40和Aβ43，但不出现Aβ42。新产生的Aβ40和Aβ43的量大致相当于Aβ46量的减少。进一步添加DAPT抑制了Aβ40/43的产生，并消除了Aβ46量的减少。这些数据表明，预先积累的Aβ46被γ-分泌酶加工成Aβ40/43，而不是Aβ42.我们试图通过LC-MS/MS鉴定根据三肽假说应从APP跨膜结构域释放的特定三肽。利用CHO细胞膜无细胞Aβ生成系统，发现在孵育过程中产生了IAT、VIV、ITL、TVI和VIT五种三肽。这些三肽的产生被L 685、458或DAPT显著抑制。这些结果有力地表明，这五个三肽是由γ-分泌酶产生的。