第十七型脊髓小腦共濟失調症 (SCA17)體外之藥物篩檢模式暨TBP與HMGB1蛋白質交互作用之研究

In vitro compound screening for SCA17 and interaction analysis between expanded TBP and HMGB1

楊淑婷(Shu-Ting Yang)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生命科學研究所/碩士(2010年)

摘要

脊髓小腦共濟失調症(SCAs)中的第1、2、3、6、7、17型和亨丁頓氏舞蹈症(HD)等神經退化性疾病，起因於特定基因上不正常的polyQ擴增，導致蛋白質的構型改變，在細胞中產生聚集(aggregation)，並透過不正常的蛋白質交互作用，影響細胞的功能。第17型SCA (SCA17)是由於位在TATA binding protein (TBP)基因上的CAG三核苷不正常擴增所導致。TBP為一轉錄起始因子，會與其他蛋白因子交互作用來調節基因的表現。TBP N端區域的polyQ長度，正常人約在25到42間，病人約在43到66間。本研究的第一目的為探討polyQ擴增是否會影響TBP和HMGB1的結合。利用原核E. coli及真核HEK293T表現的蛋白質，分別以石英晶體微量天平(QCM)與GST pull down方法，分析HMGB1與TBP蛋白間的交互作用，結果發現TBP蛋白上polyQ長度的增加，會減弱其與HMGB1蛋白的交互作用。真核HEK293T的HMGB1與TBP共表現及免疫共沉澱實驗，亦顯示TBP與HMGB1的交互作用，隨polyQ長度的增加而減弱。本研究的第二目的為藉測量thioflavin T與擴增polyQ結合產生的螢光量變化，來篩選可能抑制polyQ聚集的化合物。與剛果紅相較，所篩選的31種化合物抑制polyQ聚集的效果皆不顯著。

關鍵字

脊髓小腦共濟失調症

並列摘要

Neurodegenerative diseases such as hereditary spinocerebellar ataxias (SCAs) type 1, 2, 3, 6, 7, 17 and Huntington's disease are linked to abnormally expanded polyglutamine (polyQ) tract in the respective proteins. The polyQ expansions cause a conformational change in the polypeptide to promote misfolding and aggregation of the disease protein. The expanded polyQ protein may also acquire a toxic function through aberrant protein interactions. The disease-causing gene in SCA17 has been identified as polyQ expansion in the TATA-box binding protein (TBP) gene. TBP is a transcription initiation factor. TBP interacts with other protein factors to regulate gene expression. PolyQ length in TBP N terminal domain ranges from 25~42 in normal population and 43~66 in SCA17. The first aim of this study is to investigate if the polyQ expansion affects HMGB1 binding. By using quartz crystal microbalance (QCM) and GST pull down assay, both E. coli and HEK293T expressed proteins were used to study the interactions between HMGB1 and TBP carrying 20~61 polyQ tract. The results suggest the negative association of polyQ length and HMGB1-TBP interaction. The in vivo length-dependent negative association between TBP and HMGB1 interaction was also suggested by co-expression and co-immunoprecipitation. The second aim of this study is to screen effective chemical compounds which may inhibit polyQ aggregation using thioflavin T binding assay. Among the 31 compounds examined, none displayed effective aggregation inhibition as compared to congo red.

並列關鍵字

SCA17

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