Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP).-猫眼课题宝

淀粉样前体蛋白（APP）荧光融合蛋白的表达和加工。

DOI：

10.1016/j.bbamcr.2013.03.003

发表时间：

2013

期刊：

Biochimica et biophysica acta

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Coughlan,Kathleen;Huang,Xiangping;He,Xiangyuan;Chung,CharlotteHY;Li,Guangpu;Tang,Jordan

β-淀粉样前体蛋白（APP）在神经元中被β-和γ-分泌酶加工产生淀粉样蛋白-β（Aβ），其过度积累导致阿尔茨海默病（AD）。了解APP及其片段的亚细胞转运途径对于了解AD的发病机制具有重要意义。我们设计了包含APP（app）的C末端片段和荧光蛋白GFP（G）和DsRed（D）的融合蛋白，以允许在细胞中追踪融合蛋白和片段。表达这些蛋白质的CAD细胞发出共定位的绿色和红色荧光，并产生胞外域sGapp和sRapp以及Aβ，其水平被β和γ分泌酶抑制剂降低。通过与Rab 5共定位观察GappR在内体中的存在。这些观察结果表明，融合蛋白是膜插入的，在囊泡中转运，并通过与APP相同的机制进行蛋白水解加工。通过用放线菌酮减弱融合蛋白的合成，来自融合蛋白C-末端的单个荧光颜色出现在胞质溶胶中，该荧光颜色被β-分泌酶抑制剂强烈抑制，这表明胞外域快速离开细胞（t1/2约20分钟），而C-末端片段在细胞中保留更长时间。在活细胞中，我们观察到位于亲本融合蛋白和质膜之间的胞外域的荧光，表明这些胞外域位置是其分泌途径的一部分。我们的研究结果表明，天然胞外域并没有发挥决定性作用的APP运输和加工的关键功能和新的融合蛋白可能会导致新的见解APP的细胞内活动。

Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.