多聚谷氨酰胺长度依赖性的突变型ataxin-3片段募集野生型ataxin-3

目的脊髓小脑共济失调Ⅲ型（spinocerebellar ataxia type3,SCA3）／马查多-约瑟夫病（Machado—Joseph disease,MJD）是MJD—1基因中编码谷氨酰胺的密码子CAG的数量非正常扩增引起的一种多聚谷氨酰胺疾病,也是神经退行性疾病的一种。该病的主要病理特征为突变的ataxin-3在患者易感脑区的神经元胞核内聚集形成核内包涵体,但其致病机制和突变蛋白在核内聚集的机制仍不清楚。本研究是为了探讨突变型ataxin-3的病理学特性以及其聚集特性。方法将野生型ataxin-3及不同多聚谷氨酰胺长度的突变型ataxin-3片段共转染到人胚胎。肾细胞（HEK293 cells）和鼠成神经母细胞（neuroblastoma cells）中,用荧光显微镜观察。共转染48小时后,收集在细胞中表达的蛋白,用免疫印迹、免疫共沉淀和GST—pull down实验检测蛋白的表达及结合。结果用荧光显微镜观察到突变型ataxin-3片段在细胞内形成聚集,其聚集体上募集有野生型全长ataxin-3;突变型和野生型ataxin-3在细胞内聚集体上有共定位现象。野生型ataxin-3片段并不形成聚集体,与野生型全长ataxin-3在细胞内无聚集现象发生。免疫共沉淀技术及GST—pulldown实验显示,突变型ataxin-3片段与野生型全长ataxin-3存在相互作用,且该作用强度呈现出多聚谷氨酰胺长度依赖性。结论结果提示,野生型ataxin-3通过与突变型ataxin-3片段的相互作用沉积于突变片段形成的聚集体中,这可能影响野生型ataxin-3的正常功能,进而可能对脊髓小脑共济失调Ⅲ型（SCA3）／马查多-约瑟夫病（MJD）的发病产生影响。

PolyQ-expanded ataxin-3 interacts with full-length ataxin-3 in a polyQ length-dependent manner

Objective Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) tract in MJD-1 gene product, ataxin-3 (AT3). This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type (WT) AT3 and polyQ expanded AT3. Methods Mouse neuroblastoma (N2a) cells or HEK293 cells were co-transfected with WT AT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays. Results Using fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner. Conclusion WT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD.