Johnson G, Refolo L M, Wallace W
Molecular Geriatrics, Inc., Lake Bluff, Illinois 60044.
Ann N Y Acad Sci. 1993 Sep 24;695:194-7. doi: 10.1111/j.1749-6632.1993.tb23051.x.
The Alzheimer's disease (AD) brain contains many abnormal protein modifications. These include the abnormal processing of amyloid precursor protein (APP) to form the amyloidogenic beta/A4 peptide and the abnormal phosphorylation of tau to form A68, the major constituent of the neurofibrillary tangle. In addition, many of the biochemical alterations found in the AD brain are also found in heat-shocked or stressed cells. We used heat-shocked neuronal PC12 cells to investigate the effects of stress on APP and tau. We found that by simply exposing neuronal PC12 cells to an elevated temperature (45 degrees C) for 30 minutes, they exhibited several features characteristic of the heat shock response. These included a 45% reduction in total protein synthesis, the induction of heat shock protein (hsp) 72, and increased phosphorylation of the protein synthesis initiation factor eIF-2 alpha. The heat-shocked cells also exhibited alterations in the metabolism and phosphorylation of APP. Under heat shock conditions, we found two additional APP-like polypeptides not present in controls and a significant decrease in the phosphorylation state of APP. We also found that an A68-like protein is formed in neuronal PC12 cells when subjected to elevated temperature. This A68-like protein was formed with heat shock even in the absence of protein synthesis, suggesting that its production occurred post-translationally. The tau/A68 polypeptides were identified as phosphoproteins, and the phosphorylation of tau to form A68 was reversed with recovery of the cells from heat shock. Immunoprecipitation of lysates from heat shocked cells with antibodies to hsp72/73 resulted in co-precipitation of tau, but not A68 with hsp72 indicating a stable complex formation between these two proteins. These results suggest that heat shock proteins may play either a protective or promoting role in the formation of A68 and/or the amyloidogenic C-terminal fragment of APP.
阿尔茨海默病（AD）患者的大脑中存在许多异常的蛋白质修饰。这些修饰包括淀粉样前体蛋白（APP）异常加工形成淀粉样β/A4肽，以及tau蛋白异常磷酸化形成A68，后者是神经原纤维缠结的主要成分。此外，AD大脑中发现的许多生化改变在热休克或应激细胞中也能观察到。我们利用热休克的神经元PC12细胞来研究应激对APP和tau蛋白的影响。我们发现，只需将神经元PC12细胞在高温（45摄氏度）下暴露30分钟，它们就会表现出热休克反应的几个特征。这些特征包括总蛋白合成减少45%、诱导热休克蛋白（hsp）72以及蛋白合成起始因子eIF-2α的磷酸化增加。热休克细胞还表现出APP代谢和磷酸化的改变。在热休克条件下，我们发现了对照细胞中不存在的另外两种类似APP的多肽，并且APP的磷酸化状态显著降低。我们还发现，当神经元PC12细胞处于高温环境时会形成一种类似A68的蛋白。即使在没有蛋白质合成的情况下，热休克也会形成这种类似A68的蛋白，这表明它的产生发生在翻译后。tau/A68多肽被鉴定为磷蛋白，并且随着细胞从热休克中恢复，tau蛋白磷酸化形成A68的过程会逆转。用抗hsp72/73抗体对热休克细胞的裂解物进行免疫沉淀，结果显示tau蛋白与hsp72共沉淀，但A68不与hsp72共沉淀，这表明这两种蛋白之间形成了稳定的复合物。这些结果表明，热休克蛋白可能在A68和/或APP淀粉样生成性C末端片段的形成中发挥保护或促进作用。
