Proteomic analysis of the amyloid precursor protein fragment C99: expression in yeast.
Proteomic analysis of the amyloid precursor protein fragment C99: expression in yeast.
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DOI：
10.1016/j.ab.2007.07.033
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发表时间：
2007-11
影响因子：
2.9
通讯作者：
L. J. Sparvero;Sarah Patz;J. Brodsky;C. Coughlan
中科院分区：
文献类型：
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作者：
L. J. Sparvero;Sarah Patz;J. Brodsky;C. Coughlan
淀粉样前体蛋白 (APP) 片段的积累和聚集对于阿尔茨海默病的发展至关重要。小片段 C99 的产生被认为是 APP 加工途径中的限速步骤，这可能导致有毒 Aβ 肽的产生。也有人认为蛋白酶体有助于 APP 分解代谢。虽然许多 APP 片段的身份和聚集倾向已在体外进行了研究，但体内产生的片段的序列、结构和细胞来源仍不清楚。为了更好地识别体内产生的特定APP片段并阐明蛋白酶体在APP加工中的作用，我们开发了C99酵母表达系统。使用 Zip Tip 免疫捕获、特异性抗 Aβ 抗血清 (6E10) 和基质辅助激光解吸电离飞行时间质谱，我们在野生型酵母 (PRE1PRE2) 中鉴定了十多个 APP 生成的肽片段，在表达 C99 的蛋白酶体突变细胞 (pre1-1pre2-1) 中鉴定了三打以上的独特片段。基于免疫捕获物种的特性，我们提出蛋白酶体功能的缺陷可以通过其他蛋白酶来补偿，并且这里描述的技术组合对于进一步描述体内 APP 加工途径将是非常有价值的。
The accumulation and aggregation of fragments of amyloid precursor protein (APP) are central to the development of Alzheimer’s disease. The production of the small fragment C99 is thought to form the rate-limiting step in the APP processing pathway, which can lead to the production of the toxic Aβ peptide. It has also been suggested that the proteasome contributes to APP catabolism. While the identities and aggregation propensities of many APP fragments have been studied in vitro, the sequences, structures, and cellular sources of fragments generated in vivo remains poorly elucidated. To better identify the specific APP fragments generated in vivo and to elucidate the role of the proteasome in APP processing, we developed a C99 yeast expression system. Using Zip Tip immunocapture, a specific anti-Aβ antiserum (6E10), and matrix-assisted laser desorption ionization- time of flight mass spectrometry, we identified over one dozen APP-generated peptide fragments in wild-type yeast (PRE1PRE2) and over three dozen unique fragments in proteasome mutant cells (pre1- 1pre2-1) expressing C99. Based on the identities of the immunocaptured species, we propose that defects in proteasome function are compensated by other proteases and that the combination of techniques described here will be invaluable to further delineate the APP processing pathway in vivo.