Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene pr...Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.展开更多
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 produc...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 WTAT3 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.展开更多
伊立替康(Irinotecan,CPT-11)是一种治疗癌症的化疗药物,主要代谢产物为7-乙基-10羟基喜树碱(7-Ethyl-10-hydroxycamptothecin,SN38).课题组实验筛选结果表明伊立替康对ataxin-3核质转运有潜在影响.通过免疫荧光、微孔过滤分析以及Preto...伊立替康(Irinotecan,CPT-11)是一种治疗癌症的化疗药物,主要代谢产物为7-乙基-10羟基喜树碱(7-Ethyl-10-hydroxycamptothecin,SN38).课题组实验筛选结果表明伊立替康对ataxin-3核质转运有潜在影响.通过免疫荧光、微孔过滤分析以及PretoBlue细胞活性检测等方法,探讨伊立替康对ataxin-3质核分离、突变ataxin-3形成的聚集体以及spinocerebellar ataxia type 3(SCA3)细胞活性的影响.结果表明伊立替康在10μM处理浓度时能够减少ataxin-3进入细胞核,但对蛋白聚集体没有影响;伊立替康在0.001μM或100μM处理浓度时,对SCA3细胞具有细胞毒性作用.本研究阐释了伊立替康对SCA3细胞模型的影响,揭示了旧药新靶点,为其他神经退行性疾病的致病机理和治疗药物研发提供新依据.展开更多
基金the National Natural Sciences Foundation of China (No. 30770664)a grant from Educational Committee of Anhui Province, China (No. ZD2008008-2).
文摘Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.
基金the National Natural Sciences Foundation of China (No.30770664)a grant from Anhui Educational Committee(No. ZD2008008-2)
文摘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 WTAT3 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.
文摘伊立替康(Irinotecan,CPT-11)是一种治疗癌症的化疗药物,主要代谢产物为7-乙基-10羟基喜树碱(7-Ethyl-10-hydroxycamptothecin,SN38).课题组实验筛选结果表明伊立替康对ataxin-3核质转运有潜在影响.通过免疫荧光、微孔过滤分析以及PretoBlue细胞活性检测等方法,探讨伊立替康对ataxin-3质核分离、突变ataxin-3形成的聚集体以及spinocerebellar ataxia type 3(SCA3)细胞活性的影响.结果表明伊立替康在10μM处理浓度时能够减少ataxin-3进入细胞核,但对蛋白聚集体没有影响;伊立替康在0.001μM或100μM处理浓度时,对SCA3细胞具有细胞毒性作用.本研究阐释了伊立替康对SCA3细胞模型的影响,揭示了旧药新靶点,为其他神经退行性疾病的致病机理和治疗药物研发提供新依据.
