Study on the physiological function of synuclein family

1)在已转导α-Synudein基因的HEK293和FC12细胞中，α-Synudem被连续磷酸化。在这两种细胞系中，磷酸对磷酸酶高度敏感，因为酮酸显著稳定了磷酸盐的掺入。我们已经确定了丝氨酸129上的一个主要的磷酸化位点，该位点被酪蛋白激酶磷酸化。2)在培养的正常人星形胶质细胞中检测到α-突触核蛋白基因和蛋白的表达，免疫荧光染色显示β-突触核蛋白蛋白表达于胞浆和胞核。此外，在正常人脑组织中，星形胶质细胞中存在β-突触核蛋白免疫放射性，而少突胶质细胞中不存在。3)我们建立了爬行动物(蓝蛇)和两栖动物(牛蛙)大脑的cDNA文库，以克隆融合蛋白家族的cDNA.人β-突触核蛋白…的结构域I更多的被用来证明。从蓝绿蛇的Edna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。结果表明，这两种蛋白均与大鼠脑组织裂解液中的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之间的相互作用即使在生理条件下也存在。有趣的是，还发现在使用Cre-loxP系统的转基因小鼠中，β-突触核蛋白与金属硫蛋白结合，以阐明α-和β-突触核蛋白的生理功能。在本实验中，突触素在组织中的表达是区域特异性的，因为它是由启动子调控的。我们获得了两类转基因小鼠，Cre供体和Cre受体小鼠。将CaMK11启动子-Cre(海马)、GFAP启动子-Cre(星形胶质细胞)、Pgk2启动子-Cre(睾丸)、CAg启动子-Cre(全身)、Keratin14 Piomomomtor-Cre(皮肤)作为Cre-Dpnor转基因小鼠，以loxP-neo-loxP(Lnl)-Cz-Synudein和Lnl-α-突触核作CIE受体。较少

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