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Relationship between intracellular traffic of proteins containing expanded polyglutamine, aggregate formation, and cell death

含有扩展聚谷氨酰胺的蛋白质的细胞内运输、聚集体形成和细胞死亡之间的关系

基本信息

批准号：
10470156
负责人：
NISHIZAWA Masatoyo
金额：
$ 7.74万
依托单位：
International University of Health and Welfare (1999)Jichi Medical University (1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

In order to elucidate the mechanism of selective neuronal cell death in polyglutamine diseases caused by abnormal expansion of CAG repeat units, various constructs expressing part of ataxin-3, the product of the disease-causing gene MJD 1 of Machado-Joseph disease, as fusion protein with GFP, were introduced to COS cells, and subcellular localization of expressed ataxin-3 fragments was analyzed under fluorescence microscopy.We found that the C-terminal end of ataxin-3 is essential to formation of cytoplasmic aggregates. These cytoplasmic aggregates were surrounding the nucleus and formed mostly in the juxtanuclear region. Confocal laser microscopy revealed that these aggregates were co-localized with tubulin, and especially, juxtanuclear aggregates were co-localized with gamma-tubulin, which is now known as a marker of microtubulus organizing center (MTOC). These results indicate that the C-terminal portion of ataxin-3 can interact with tubulins, and the aggregates formed by expanded polyglutamine and the C-terminal region, accumulate in the MTOCs.The N-terminal region flanking the polyglutamine tract of ataxin-3 contained active sequence of nuclear localization signal and also a region with coiled-coil conformation. The latter region was found to be invloved in the aggregates formed by expanded polyglutamine. Overexpression of the construct expressing the latter region resulted in the reduction of intranuclear aggregates formed by expanded polyglutamine derived from DRPLA protein and subsequent cell death.Our present study indicates the important role of intramolecular domains of the products of disease-causig genes in the formation of cytoplasmic and intranuclear aggregates.

为了阐明CAG重复单元异常扩增引起的多聚谷氨酰胺疾病中选择性神经元细胞死亡的机制，将Machado-Joseph病致病基因MJD - 1的产物ataxin-3部分表达的不同构建体作为GFP融合蛋白引入COS细胞，在荧光显微镜下分析表达的ataxin-3片段的亚细胞定位。我们发现ataxin-3的c末端对细胞质聚集体的形成至关重要。这些细胞质聚集体围绕着细胞核，主要形成于核旁区。激光共聚焦显微镜显示，这些聚集体与微管蛋白共定位，特别是近核聚集体与γ -微管蛋白共定位，γ -微管蛋白是微管组织中心（MTOC）的标志。这些结果表明，ataxin-3的c端部分可以与微管蛋白相互作用，并且由扩展的聚谷氨酰胺和c端区域形成的聚集体在mtoc中积累。在ataxin-3聚谷氨酰胺通道两侧的n端区域含有核定位信号的活性序列，也有一个螺旋状构象区域。后一区域被发现参与膨胀聚谷氨酰胺形成的聚集体。表达后一区域的构建体的过表达导致由DRPLA蛋白衍生的聚谷氨酰胺形成的核内聚集体减少和随后的细胞死亡。我们目前的研究表明致病基因产物的分子内结构域在细胞质和核内聚集体形成中的重要作用。