Interaction Analysis Between Polyglutamine-expanded TATA Box Binding Protein (TBP) and High Mobility Group Box 1 (HMGB1)

多麩醯胺擴增TBP與HMGB1的交互作用研究

10.6248/BF.2013.48.03

楊淑婷(Shu-Ting Yang)；陳襄銘(Hsiang-Ming Chen)；李麗卿(Li-Ching Lee)；黃詩涵(Shih-Han Huang)；林志信(Chih-Hsin Lin)；李冠群(Guan-Chiun Lee)；李振綱(Cheng-Kang Lee)；李桂楨(Guey-Jen Lee-Chen)

免疫共沉澱 ； 石英晶體微量天平 ； TBP ； HMGB1 ； co-IP ； GST pull-down ； QCM

Bio Formosa

48卷（2013 / 06 / 01）

17 - 26

英文

第十七型脊髓小腦共濟失調症（SCA17）與轉錄起始因子TATA binding protein （TBP）基因上的多麩醯胺擴增相關。TBP與包括high mobility group box 1 （HMGB1）在內的其他蛋白因子交互作用，來調節基因的表現。本研究藉重組的HMGB1 及TBP／Q20～61 N端蛋白，探討TBP Q長度是否影響其與HMGB1的結合。首先共表現HMGB1及TBP於 HEK-293細胞後，利用免疫共沉澱及GST pull-down試驗，確認HMGB1 與TBP在細胞內的結合。其次原核E. coli表現的重組HMGB1 及TBP／Q20～61蛋白的GSTpull-down試驗，亦顯示HMGB1與TBP的結合。最後即時石英晶體微量天平（QCM）試驗顯示，HMGB1-TBP交互作用隨TBP蛋白上polyQ長度的增加而減弱。綜合先前報導的HMGB1-TBP交互作用可增強TBP結合TATA速率及穩定性，我們的研究顯示多麩醯胺擴增TBP與HMGB1結合的減弱，可能與SCA17致病機轉相關。

Spinocerebellar ataxia type 17 (SCA17) involves the expression of a polyglutamine-expanded TATA box binding protein (TBP), a general transcription initiation factor. TBP interacts with other protein factors, including high mobility group box 1 (HMGB1), to regulate gene expression. In this study, we examined the effect of TBP Q-tract length on HMGB1 binding using recombinant HMGB1 and N-terminal TBP/Q20~61 proteins. The intracellular association of HMGB1 with TBP was first confirmed by using a half-in vivo co-immunoprecipitation (co-IP) and glutathione S-transferase (GST) pull-down assays in HMGB1 and TBP co-expressed HEK-293 cells. GST pull-down assay using soluble recombinant HMGB1 and TBP/Q20~61 proteins expressed in E. coli also revealed the binding activity between recombinant HMGB1 and TBP. When the binding strength was explored by using a real time quartz crystal microbalance (QCM) assay, a Q-tract length-dependent decrease of HMGB1-TBP interaction was demonstrated. Together with the reported HMGB1-TBP interaction to increase the rate of TBP binding and the stability of the TBP/TATA interaction, our study results suggest that decrease HMGB1 binding to polyQ-expanded TBP may contribute to the pathogenesis of SCA17.