Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.
PLoS One. 2012;7(4):e35302. doi: 10.1371/journal.pone.0035302. Epub 2012 Apr 17.
Spinocerebellar ataxia 17 (SCA17) is caused by expansion of the polyglutamine (polyQ) tract in human TATA-box binding protein (TBP) that is ubiquitously expressed in both central nervous system and peripheral tissues. The spectrum of SCA17 clinical presentation is broad. The precise pathogenic mechanism in SCA17 remains unclear. Previously proteomics study using a cellular model of SCA17 has revealed reduced expression of heat shock 70 kDa protein 5 (HSPA5) and heat shock 70 kDa protein 8 (HSPA8), suggesting that impaired protein folding may contribute to the cell dysfunction of SCA17 (Lee et al., 2009). In lymphoblastoid cells, HSPA5 and HSPA8 expression levels in cells with mutant TBP were also significantly lower than that of the control cells (Chen et al., 2010). As nuclear transcription factor Y (NFY) has been reported to regulate HSPA5 transcription, we focused on if NFY activity and HSPA5 expression in SCA17 cells are altered. Here, we show that TBP interacts with NFY subunit A (NFYA) in HEK-293 cells and NFYA incorporated into mutant TBP aggregates. In both HEK-293 and SH-SY5Y cells expressing TBP/Q(61~79), the level of soluble NFYA was significantly reduced. In vitro binding assay revealed that the interaction between TBP and NFYA is direct. HSPA5 luciferase reporter assay and endogenous HSPA5 expression analysis in NFYA cDNA and siRNA transfection cells further clarified the important role of NFYA in regulating HSPA5 transcription. In SCA17 cells, HSPA5 promoter activity was activated as a compensatory response before aggregate formation. NFYA dysfunction was indicated in SCA17 cells as HSPA5 promoter activity reduced along with TBP aggregate formation. Because essential roles of HSPA5 in protection from neuronal apoptosis have been shown in a mouse model, NFYA could be a target of mutant TBP in SCA17.
脊髓小脑性共济失调 17 型（SCA17）是由人类 TATA 框结合蛋白（TBP）中多聚谷氨酰胺（polyQ）片段的扩展引起的，该蛋白在中枢神经系统和外周组织中广泛表达。SCA17 的临床表现谱很广。SCA17 的精确发病机制尚不清楚。以前使用 SCA17 的细胞模型进行的蛋白质组学研究表明，热休克 70kDa 蛋白 5（HSPA5）和热休克 70kDa 蛋白 8（HSPA8）的表达降低，表明蛋白质折叠受损可能导致 SCA17 的细胞功能障碍（Lee 等人，2009）。在淋巴母细胞样细胞中，突变 TBP 细胞中的 HSPA5 和 HSPA8 表达水平也明显低于对照细胞（Chen 等人，2010）。由于核转录因子 Y（NFY）已被报道调节 HSPA5 转录，我们专注于 SCA17 细胞中的 NFY 活性和 HSPA5 表达是否改变。在这里，我们表明 TBP 在 HEK-293 细胞中与 NFY 亚基 A（NFYA）相互作用，并且 NFYA 整合到突变 TBP 聚集体中。在表达 TBP/Q（61~79）的 HEK-293 和 SH-SY5Y 细胞中，可溶性 NFYA 的水平显着降低。体外结合实验表明 TBP 与 NFYA 的相互作用是直接的。HSPA5 荧光素酶报告基因测定和 NFYA cDNA 和 siRNA 转染细胞中的内源性 HSPA5 表达分析进一步阐明了 NFYA 在调节 HSPA5 转录中的重要作用。在 SCA17 细胞中，在聚集形成之前，HSPA5 启动子活性被激活作为代偿反应。在 SCA17 细胞中，NFYA 功能障碍表明随着 TBP 聚集体形成，HSPA5 启动子活性降低。因为 HSPA5 在保护神经元凋亡方面的重要作用在小鼠模型中已经得到证实，NFYA 可能是 SCA17 中突变 TBP 的靶标。
