Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes
Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes
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DOI：
10.1242/jcs.00643
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发表时间：
2003-08-15
影响因子：
4
通讯作者：
Hyman, BT
中科院分区：
文献类型：
--
作者：
Kinoshita, A;Fukumoto, H;Hyman, BT
淀粉样β肽是由淀粉样前体蛋白（APP）经蛋白水解加工而得，在阿尔茨海默病的老年斑中积累。β-分泌酶（Asp 2）在淀粉样蛋白-β的N-末端切割APP，最近被鉴定为蛋白酶BACE。在本研究中，我们研究了APP和BACE之间的相互作用的亚细胞定位通过使用双重免疫荧光和荧光共振能量转移（FRET）的方法。细胞表面APP和BACE，研究通过使用针对其胞外域在活的H4神经胶质瘤细胞共转染APP和BACE的抗体，显示精致的共定位，并表现出非常密切的相互作用，通过FRET分析。大多数细胞表面APP和BACE在15分钟后被内化，但它们在早期内体区室中保持强烈的共定位在一起，其中FRET分析证明了持续的密切相互作用。相比之下，在稍后的时间点，几乎没有共定位或FRET中观察到溶酶体隔室。为了确定细胞表面和内体上的APP-BACE相互作用是否有助于淀粉样蛋白-β的合成，我们标记了细胞表面APP，并在30分钟内证明了标记的淀粉样蛋白-β的可检测水平。APP-Swedish突变蛋白增强了细胞表面APP的β淀粉样蛋白合成，这与其是比野生型APP更好的BACE底物的观察结果一致。综上所述，这些数据证实了早期内体中APP-BACE的密切相互作用，并突出了细胞表面作为APP-BACE相互作用的额外潜在位点。
Amyloid-beta peptide, which accumulates in senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolytic processing. beta-secretase (Asp2), which cleaves APP at the N-terminus of amyloid-beta, has recently been identified to be the protease BACE. In the present study, we examined the subcellular localization of interactions between APP and BACE by using both double immunofluorescence and a fluorescence resonance energy transfer (FRET) approach. Cell surface APP and BACE, studied by using antibodies directed against their ectodomains in living H4 neuroglioma cells co-transfected with APP and BACE, showed exquisite co-localization and demonstrated a very close interaction by FRET analysis. The majority of cell surface APP and BACE were internalized after 15 minutes, but they remained strongly co-localized together in the early endosomal compartment, where FRET analysis demonstrated a continued close interaction. By contrast, at later timepoints, almost no co-localization or FRET was observed in lysosomal compartments. To determine whether the APP-BACE interaction on cell surface and endosomes contributed to amyloid-beta synthesis, we labeled cell surface APP and demonstrated detectable levels of labeled amyloid-beta within 30 minutes. APP-Swedish mutant protein enhanced amyloid-beta synthesis from cell surface APP, consistent with the observation that it is a better BACE substrate than wild-type APP. Taken together, these data confirm a close APP-BACE interaction in early endosomes, and highlight the cell surface as an additional potential site of APP-BACE interaction.