14-3-3蛋白在阿尔茨海默病SET核运输中的作用及其机理研究

PP2A inactivation plays a pivotal role in abnormal tau phosphorylation and AD pathogenesis. Aberrant cytoplasmic localization of nuclear protein SET is crucial to PP2A inactivation, yet the mechanism is unknown. Meanwhile, 14-3-3 is highly expressed in the AD brain, a protein known for negative nuclear import function. Previously we have found that co-transfection of SET and 14-3-3 leads to SET cytoplasmic retention, significant increase of their binding, and enhanced tau hyperphosphorylation. Current project aims at: 1.Investigating 14-3-3 in interacting with SET and nuclear import proteins, its role in the phosphorylation of SET nuclear localization signal; determining the intervention effects of 14-3-3 in the formation SET nuclear import complex; clarifying the molecular mechanism of SET cytoplasmic retention caused by 14-3-3. 2.Generating site-directed 14-3-3 mutants which may bind to the presumed SET serine interacting domain, thus determining the exact binding sites; synthesizing SET serine domain-binding 14-3-3 peptides, analyzing their intervention effects in repressing SET cytoplasmic retention and in the AD-like pathogenesis. The expecting results will provide new clues to the AD pathogenesis and provide molecular drug targets for prevention and treatment.

PP2A失活在引起tau异常磷酸化和AD发病中起关键作用，核蛋白SET于胞浆异常定位是PP2A失活的重要原因，但SET滞留胞浆的机制不明。同时，具有核输入负调功能的14-3-3蛋白在AD脑内亦高表达。申请人研究发现：SET与14-3-3蛋白共转染导致SET滞留于胞浆且二者结合显著增加，并加重tau过度磷酸化。在此基础上，本项目拟1.探讨14-3-3蛋白对SET与核输入蛋白相互作用及对SET核定位信号磷酸化修饰的影响，明确14-3-3蛋白对SET核输入复合物形成的干预作用，阐明14-3-3蛋白致SET滞留胞浆的分子机制；2.分别应用基因定点突变14-3-3蛋白与SET推定蛋白结合域序列中的丝氨酸和合成该蛋白结合域肽段干预，确定SET与14-3-3蛋白结合功能位点，探讨合成肽阻遏SET滞留胞浆的作用及对AD样病理学变化的影响。本项目研究结果将为AD发病机制提供新线索并为其防治提供分子药靶。

PP2A活性下降导致tau过度磷酸化，进而促进神经原纤维缠结(neurofibrillary tangles, NFTs)形成，是阿尔茨海默病(Alzheimer disease, AD)发病的关键环节。核蛋白SET是体内PP2A特异、高效的抑制剂。AD患者脑内 SET水平显著增高，从核转运至胞浆聚集，并发现与神经元胞浆内PP2A及异常过度磷酸化的tau蛋白共定位。14-3-3蛋白作为接头/支架蛋白参与核运输中的调控作用。本项目调查了14-3-3蛋白对SET核运输的影响及其参与AD发病的相关分子机制。我们发现：14-3-3β/ζ可以通过与SET蛋白的NESGDPSSKST序列结合，导致SET与importinα结合显著减少，从而引起SET核输入障碍，滞留胞浆，显著抑制PP2A活性，并导致tau蛋白发生过度磷酸化，神经元树突损伤，LTP及学习和记忆的损伤。通过给予Tat-NESGDPSSKST肽段可以通过有效竞争性结合14-3-3β/ζ来释放SET，使SET核输入正常而主要定位与胞核，减少对胞浆中PP2A的抑制，恢复PP2A活性，减少神经元树突损伤，恢复LTP及学习和记忆的损伤等。