DNMT1核运输障碍在阿尔茨海默病发病中的作用及其机制研究

In AD brains, nuclear protein DNMT1, which plays a crucial role in the methylation of tau-related gene GSK-3β and Aβ-generation-related genes, exhibited quantitative deficits in transport into the nucleus and aberrant cytoplasmic localization, yet the mechanism is unknown. Meanwhile, 14-3-3 is highly expressed in the AD brains, a protein known for regulating nuclear import negatively. By protein BLAST, DNMT1 contains a sequence, which is consistent coincidentally with 14-3-3 consensus binding motif. Previously, we have found that overexpression of 14-3-3β/ζ induced cytoplasmic detention of endogenous DNMT1, which co-localized with 14-3-3β/ζ. Current project aims at: 1. Investigating the interaction of 14-3-3 with DNMT1 and importin proteins, and its role in the phosphorylation of DNMT1 nuclear localization signal; exploring the molecular mechanism by which 14-3-3 intervenes in DNMT1 nuclear import; 2. Generating site-directed DNMT1 nuclear localization signal mutants, thus detecting its mutants subcellular distribution and their interaction with importin proteins; clarifying the NLS of DNMT1 and the mechanism by which NLS abnormal phosphorylation induced DNMT1 cytoplasmic detention; 3. Determining the role of DNMT1 cytoplasmic detention in the pathogenesis of AD in vitro and in vivo, and analyzing the protection of TBB on AD-like pathology. The expecting results will provide new clues to AD pathogenesis and molecular drug targets for prevention and treatment of AD.

对tau激酶GSK-3β和Aβ生成相关基因的甲基化修饰至关重要的核蛋白DNMT1在AD患者脑内大量聚集于胞浆，但分子机制不明。通过序列比对，DNMT1中含有能与亦在AD脑内高表达的核输入负调蛋白14-3-3结合的区域。申请者研究发现：过表达14-3-3β/ζ使内源性DNMT1在胞浆中大量聚集，且与14-3-3共定位。在此基础上，本项目拟进一步1．检测14-3-3干预下与DNMT1的结合以及对核定位信号中丝氨酸的磷酸化修饰，探讨14-3-3负调DNMT1核输入的作用机制；2．定点突变DNMT1核定位信号中丝氨酸和碱性氨基酸，检测各突变体的亚细胞定位以及与核输入蛋白的相互作用，阐明NLS异常磷酸化导致DNMT1核运输障碍的机制；3．研究DNMT1核运输障碍在AD发病中的作用，并探讨外源性抑制DNMT1磷酸化对AD样神经病变和学习记忆障碍的改善作用。本项目将为AD防治提供新的分子靶点和实验模型。