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Study on the physiological function of synuclein family

突触核蛋白家族的生理功能研究

基本信息

批准号：
11557196
负责人：
NAKAJO Shigeo
金额：
$ 6.98万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557196/
关键词：
synuclein synuclein-binding protein neuro-degeneration transgenic mouse 14-3-3 metallothionein 14-3-3θ PNP14 神経変性疾患

项目摘要

1) It was found that a-synudem is consiturtively phosphoiylated in both cells HEK 293 and FC12 which had been previously transfected with α-synudein gene. In both cell lines phosphorylalion was highly sensitive to phosphatases, since okedaic acid markedly stabilized phosphate incorporation. We have identified a major phosphorylation site at serine 129 which is phosphoiylated by casein kinases. It may be indicated that the function of α-synuciein is regulated by phosphorylation/dephosphorylation reaction.2) β-synuclein mRNA and protein were detected in normal human astrocytes in culture, and immunofluorescent staining showed that β-synuclein protein was expressed within the cytoplasm and nudeus. Furthermore, β-synuclein immunoreadivity was present in astrocytes, but not in oligodendrocytes, in normal human brain tissues.3) We have prqared cDNA library fmm the cerebrum of reptiles (blue green snake) and amphibian (bullfrog) to clone cDNA of synudein family. Domain I of human β-synuclein … More was used as a prove. Ten positive clones have been obtained from the eDNA library of blue green snake. The sequence analysis is underadvance,4) Using yeast two-hybrid system, we have identified 14-3-30 and metallothionein2A as β-synudein-binding protein. It has been shown that both proteins interact with β-synuclein in rat brain lysate, indicating that those interactions between β-synuclein and 14-3-30 and metallothionein2A occure even under the physiological conditions. Interestingly, it was also shown that β-synuclein bound to metallothionein3.5) Transgenic mice using Cre-loxp system were produced to clarify the physiological function of α- and β-synucleins. In this experiment expression of synudeins is region specific in tissue since it is regulated by promotor used. We produced transgenic mice of two category, Cre-donor and Cre-acceptor mice. CaMkll promotor-Cre (hippocampus), GFAP promotor-Cre (astrocytes), Pgk2 promotor-Cre (testes), CAG promoter-Cre (whole body), Keratin14 piomotor-Cre (skin) transgenic mice were produced as Cre-dpnor, and loxP-neo-loxP (LNL)-cz-synudein and LNL-α-synuclein mice were as Cie-acceptor. Less

1)结果表明，在已转染α-synudein基因的HEK 293和FC 12细胞中，α-synudem均发生结构性磷酸化。在这两种细胞系中，磷酸化酶是高度敏感的磷酸酶，因为okedaic酸显着稳定磷酸盐掺入。我们已经确定了一个主要的磷酸化位点在丝氨酸129，这是磷酸化的酪蛋白激酶。2）在培养的正常人星形胶质细胞中检测到β-synuclein mRNA和蛋白的表达，免疫荧光染色显示β-synuclein蛋白在细胞质和细胞核中均有表达。3）从爬行类动物（蓝绿色蛇）和两栖类动物（牛蛙）大脑中构建了synudein家族cDNA文库。人β-突触核蛋白结构域I ...更多信息被用作证明。从蓝绿色蛇cDNA文库中获得10个阳性克隆。4）利用酵母双杂交系统，我们鉴定了14-3-30和金属硫蛋白2A为β-synudein结合蛋白。在大鼠脑裂解物中，这两种蛋白质都与β-突触核蛋白相互作用，表明β-突触核蛋白与14-3-30和金属硫蛋白2A之间的相互作用甚至在生理条件下也发生。有趣的是，还显示β-突触核蛋白与金属硫蛋白结合3.5）使用Cre-loxp系统产生转基因小鼠以阐明α-和β-突触核蛋白的生理功能。在本实验中，synudeins的表达是区域特异性的，因为它是由所使用的启动子调控的。我们生产了两类转基因小鼠，Cre供体和Cre受体小鼠。以CaMk 11启动子-Cre（海马）、GFAP启动子-Cre（星形胶质细胞）、Pgk 2启动子-Cre（睾丸）、CAG启动子-Cre（全身）、Keratin 14启动子-Cre（皮肤）转基因小鼠作为Cre-dpnor，loxP-neo-loxP（LNL）-cz-synudein和LNL-α-synuclein小鼠作为Cie受体。少