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The mechanisms of aggregation, accumulation and degradation of the genetically causative protein related to familial dementia diseases

家族性痴呆疾病相关遗传致病蛋白的聚集、积累和降解机制

基本信息

批准号：
21390333
负责人：
TAKEDA Masatoshi
金额：
$ 11.56万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

项目摘要

Tau protein is hyperphosphorylated, ubiquitinated and accumulated in FTDP-17 brains, however mechanisms of the accumulation is still unclear. To understand the involvement of proteases in metabolisms of tau protein, some of protease inhibitors were employed in cultured cells. Pulse-chase experiments revealed proteolysis of tau protein was attenuated when treated with puromycin. And increased tau protein levels were also observed in cells treated with siRNA to puromycin-sensitive aminopeptidase(PSA) for inhibition of its expression. Those data suggest that PSA is a protease which regulates proteolysis of tau predominantly in cells. The protein metabolism of tau containing FTDP-17 mutations was also investigated employing pulse-chase experiments and attenuated proteolysis of tau was observed in the cells transfected with the mutant tau. Phosphorylation of tau at Thr231, Ser396 and Ser409 was increased in the cells transfected with V337M, R406W, and R406W mutant tau gene, respectively. These results suggest that attenuated proteolysis of FTDP-17 mutant tau might be explained by the increased phosphorylation levels resulting in resistance to proteolysis. Furthermore, in cells treated with fumagilin, an inhibitor to methionine aminopeptidase, tau protein with N-terminal methionine increased similar to puromicin-treated cells, suggesting that aminoterminal processing of tau protein is important for tau degradation.

Tau蛋白在FTDP-17脑中过度磷酸化、泛素化并积累，但其积累机制尚不清楚。为了了解蛋白酶参与tau蛋白的代谢，在培养的细胞中使用一些蛋白酶抑制剂。脉冲追踪实验表明，用嘌呤霉素处理时，tau蛋白的蛋白水解减弱。在用针对嘌呤霉素敏感性氨肽酶（PSA）的siRNA抑制其表达的细胞中也观察到tau蛋白水平增加。这些数据表明，PSA是一种蛋白酶，主要在细胞中调节tau的蛋白水解。还采用脉冲追踪实验研究了含有FTDP-17突变的tau的蛋白质代谢，并且在用突变体tau转染的细胞中观察到tau的减弱的蛋白质水解。V337 M、R406 W和R406 W突变体tau基因转染细胞后，tau蛋白在Thr 231、Ser 396和Ser 409位点的磷酸化水平分别升高。这些结果表明，FTDP-17突变tau蛋白水解减弱可能是由于磷酸化水平增加导致蛋白水解抗性。此外，在用烟曲霉素（甲硫氨酸氨基肽酶的抑制剂）处理的细胞中，具有N-末端甲硫氨酸的tau蛋白增加类似于嘌呤霉素处理的细胞，表明tau蛋白的氨基末端加工对于tau降解是重要的。