臺灣博碩士論文加值系統

以酵母菌之雙雜交系統, 用人類巨細胞病毒迅早期蛋白IE2之C端作餌, 找出相互作用蛋白Nrf1, 此蛋白屬於CNC-bZIP蛋白家族成員. 在CNC-bZIP蛋白家族成員中的Nrf1和Nrf2, 皆和IE2具特異性結和能力. Nrf1胺基酸序列331至448包含DNA鍵結區及雙元複合體 (dimer) 區足以和IE2作用. 此相互作用藉由in vitro的GST-pull down和in vivo的共同沉澱 (coimmunoprecipitation) 得到進一步證實. 在轉染研究中帶六個NF-E2 DNA連結序列的訊息轉錄能被IE2所抑制, 並且IE2也降低由LexA和CNC-bZIP蛋白家族所合成之複合蛋白對帶有LexA reporter之訊息轉錄. 更重要的是IE2對Nrf1/Maf異複體和NF-E2 DNA連結序列之鍵結沒什影響, 此顯示CNC-bZIP蛋白家族和IE2連結是可能的機轉. 同時, Nrf1抑制IE2的自我調節能力, 此抑制作用可由單獨的Nrf1及Nrf1/Maf複合體抑制IE2 DNA連結能力來解釋, 由上得知IE2和CNC蛋白家族具相互拮抗作用.

Using the C-terminal region of the HCMV IE2 protein as bait in the yeast two-hybrid system, Nrf1, a member of the CNC-bZIP family, was isolated. Among members of the CNC-bZIP family tested in yeast, Nrf1 and, to a lesser extent, Nrf2 specifically interact with IE2. Residues 331-448 encompassing the DNA-binding and the dimerization domains of Nrf1 are sufficient for interaction with IE2. The interaction was further confirmed in vitro by a GST pull-down assay and in vivo by co-immunoprecipitation. In transient transfection studies, transcription driven by six copies of an NF-E2 site, a cognate site for a heterodimer between members of the CNC-bZIP and the small Maf families, is repressed by IE2. Furthermore, IE2 down-regulates transcription from a LexA reporter driven by chimeric proteins between the DNA-binding domain of LexA and members of the CNC-bZIP family. Importantly, IE2 appears to have little effect on the binding of the Nrf1/MafK heterodimer to an NF-E2 site, suggesting that recruitment of IE2 to the promoter by CNC-bZIP factors is the underlying mechanism for the inhibition. In a parallel study, Nrf1 attenuates the negative autoregulation of IE2. The attenuation could be explained by the finding that Nrf1 functions alone and synergistically with its heterodimerization partner, MafK, in inhibiting the DNA-binding activity of IE2. Taken together, these results demonstrate the mutual antagonism between IE2 and members of the CNC-bZIP family.
Contents…………………………………………………………………I
List of Figures…………………………………………………………..II
List of Abbreviations…………………………………………………...IV
Chinese Abstract……………………………………………………….V
English Abstract………………………………………………………..VI
Introduction…………………………………………………………….1-28
Materials and methods…………………………………………………29-42
Results……………………………………………………………….…43-52
Discussion……………………………………………………………...53-58
References……………………………………………………………..59-89
Figures…………………………………………………………………90-110
Appendix

Cover
Contents
List of Figures
List of Abbreviations
Chinese Abstract
English Abstract
Introduction
Materials and methods
Results
Discussion
References
Figures
Appendix

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