Lee Li-Ching, Chen Chiung-Mei, Chen Fen-Lin, Lin Pei-Ying, Hsiao Ya-Chin, Wang Pin-Rong, Su Ming-Tsan, Hsieh-Li Hsiu-Mei, Hwang Ji-Chuu, Wu Chung-Hsin, Lee Guan-Chiun, Singh Sher, Lin Yenshou, Hsieh Sen-Yung, Lee-Chen Guey-Jen, Lin Jung-Yaw
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.
Clin Chim Acta. 2009 Feb;400(1-2):56-62. doi: 10.1016/j.cca.2008.10.013. Epub 2008 Oct 30.
Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous system and peripheral tissues. The underlying molecular changes of SCA17 are rarely explored.
To study the molecular mechanisms underlying SCA17, we generated stably induced isogenic 293 cells expressing normal TBP-Q(36) and expanded TBP-Q(61) and analyzed the expressed proteins using two-dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting.
Upon induction with doxycycline, the expanded TBP-Q(61) formed aggregates with significant increase in the cell population at subG1 phase and cleaved caspase-3. Proteomics study identified a total of 16 proteins with expression changes greater than 1.5 fold. Among the 16 proteins, PARK7, GLRX3, HNRNPA1, GINS1, ENO1, HNRPK and NPM1 are increased, and SERPINA5, HSPA5, VCL, KHSRP, HSPA8, HNRPH1, IMMT, VCP and HNRNPL are decreased in cells expressing TBP-Q(61) compared with those expressing TBP-Q(36). The altered expression of HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses.
The results illustrate the utility of proteomics to identify alterations of proteins which underlie pathogenesis of SCA17, and may serve as potential therapeutic targets.
已确定TATA盒结合蛋白（TBP）基因的CAG重复序列扩增是脊髓小脑共济失调17型（SCA17）的致病突变。TBP在中枢神经系统和外周组织中均广泛表达。SCA17潜在的分子变化鲜有研究。
为研究SCA17潜在的分子机制，我们构建了稳定诱导的同基因293细胞，分别表达正常的TBP-Q(36)和扩增的TBP-Q(61)，并使用二维差异凝胶电泳（2D-DIGE）分析表达的蛋白质，随后进行质谱分析和免疫印迹。
用强力霉素诱导后，扩增的TBP-Q(61)形成聚集体，亚G1期细胞群体和裂解的半胱天冬酶-3显著增加。蛋白质组学研究共鉴定出16种表达变化大于1.5倍的蛋白质。在这16种蛋白质中，与表达TBP-Q(36)的细胞相比，表达TBP-Q(61)的细胞中PARK7、GLRX3、HNRNPA1、GINS1、ENO1、HNRPK和NPM1增加，而SERPINA5、HSPA5、VCL、KHSRP、HSPA8、HNRPH1、IMMT、VCP和HNRNPL减少。HSPA5、HSPA8和PARK7表达的改变通过二维和Western免疫印迹分析进一步得到验证。
结果表明蛋白质组学可用于鉴定构成SCA17发病机制基础的蛋白质改变，这些蛋白质可能作为潜在的治疗靶点。
