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

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，将表达部分共济失调蛋白-3（Machado-Joseph病的致病基因MJD 1的产物）的各种构建体作为与GFP的融合蛋白引入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，发现ataxin-3的C末端是胞质聚集体形成的关键。这些细胞质聚集体围绕着细胞核，主要形成于胞核周围。共聚焦激光显微镜显示，这些聚集体与微管蛋白共定位，特别是，微管核聚集体与γ-微管蛋白共定位，γ-微管蛋白现在被认为是微管组织中心（MTOC）的标志物。这些结果表明，ataxin-3的C端部分可与微管蛋白相互作用，并在MTOCs中聚集由多聚谷氨酰胺和C端区域形成的聚集体，ataxin-3多聚谷氨酰胺束两侧的N端区域含有核定位信号的活性序列和卷曲螺旋构象。后者被发现是invloved在聚集体所形成的扩展聚谷氨酰胺。表达后一个区域的构建体的过表达导致由来自DRPLA蛋白的扩展聚谷氨酰胺形成的核内聚集体的减少和随后的细胞death.Our本研究表明疾病causig基因产物的分子内结构域在细胞质和核内聚集体的形成中的重要作用。

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.