文档介绍：Ac糖基转移酶表达下调对tau蛋白磷酸化的影响
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【摘要】以人OAc糖基转移酶(OGT)基因为靶点,研究小干扰RNA(small interference RNA,siRNA)对HEK293T细胞中OGT基因表达的抑制作用,以及这种抑制作用对tau蛋白磷酸化与糖基化修饰的影响。方法: 根据人OGT基因序列特点,设计高效且特异性强的siRNA。用脂质体转染siRNA进入HEK293T细胞,通过实时PCR检测siRNA对OGT基因表达的抑制。用蛋白免疫印迹法检测tau蛋白糖基化与磷酸化修饰的变化。结果: 设计的siRNA能够有效抑制OGT基因的表达,与空白组相比,%左右。OGT基因表达的下调使tau蛋白糖基化水平下降,而磷酸化水平增高。结论: tau蛋白的糖基化负调节其磷酸化,葡萄糖摄入减少或代谢降低可能在阿尔茨海默尔病的发生发展中起关键作用。
【关键词】阿尔茨海默尔病 tau蛋白 OAc糖基转移酶 RNA干扰
[Abstract] Objective: To investigate the inhibitory effect of small interference RNA(siRNA)targeting OGT gene on the alteration of tau phosphorylation and glycosylation level in human being. Methods: The siRNA
NC and the siRNA targeting OGT gene were chemically synthesized and transfected into HEK293T cells via time PCR was employed to evaluate the efficacy of RNA interference. The level of tau phosphorylation and glycosylation were detected by Western blot. Results: The designed siRNA could effectivly downregulate the OGT mRNA expression to % pared with blank group. The level of the tau phosphorylation at various sites was significantly upregulated and the level of the tau glycosylation was downregulated with the inhibition of OGT gene expression. Conclusion: The modification of phosphorylation and glycosylation of tau protein indicated the apparent negative correlation, which probably was induced by deficient brain glucose uptake/metabolism in Alzheimer′s disease
[Key words] Alzheimer′s disease; tau; OGT; RNA interfering
阿尔茨海默尔病(Alzheimer′s disease, AD)的病理特征包括大脑局部尤其是海马和皮层神经元退行性病变,细胞内神经原纤维缠结(neurofibrillary tangles,NFT)和细胞外老年斑(senile plaque ,SP)沉积。其中NFT的数量和患者的痴呆程度呈明显的正相关,被认为是AD患者神经原退化的病理基础[1,2]。NFT的主要成分是由异常过度磷酸化tau蛋白聚集而形成的双螺旋丝(paired helical filaments,PHFs)。过度磷酸化的tau蛋白除失去了其刺激和促进微管组装的功能外,还变成了毒性分子,可猎获正常的微管相关蛋白,使得微管崩解,神经细胞退化,最终导致AD患者的认知功能受到严重的损害。
蛋白质的修饰方式主要有磷酸化、糖基化、泛素化、经典糖基化、硝基化和O
Ac糖基化。其中蛋白质OAc 糖基化是发生在细胞质与细胞核内的一种广泛动态的蛋白质翻译后修饰现象。这种糖基化是单个N乙酰氨基葡萄糖(Nacetylglucosamine