Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27176
Title: | mLRH-1 結構功能之探討:PIASy 作用區、Hinge 結構區及核定位訊號 Structure-function relationship of mLRH-1 : PIASy interaction region, hinge and NLS domains |
Authors: | Chih-Hung Wang 王志宏 |
Advisor: | 胡孟君 |
Keyword: | 抑制機制,基因調控,核定位訊號,交互作用, LRH-1,PIASy,interaction,NLS, |
Publication Year : | 2008 |
Degree: | 碩士 |
Abstract: | 肝受器同源體-1 (Liver receptor homolog-1 ; LRH-1) 為核受器 NR5A 家族的成員之一,由於目前仍然未發現其配體,因此稱之為孤兒核受器。LRH-1 主要表現於肝腸組織與卵巢。已知 LRH-1 具有調控類固醇生成基因的能力,本實驗室先前已證實 mLRH-1 可以促進類固醇生成基因 CYP11A1 啟動子的轉錄活性,並發現此作用明顯地會被 PIASy 所抑制,本論文即探討 PIASy 抑制mLRH-1 轉錄活性的分子機制。為了找出 mLRH-1 與 PIASy 交互作用區域,我們建構了不同片段的 Gal4-mLRH-1以及 VP16-PIASy 融合蛋白,並利用哺乳類雙雜交系統進行分析。結果發現 mLRH-1 是利用 C 端胺基酸 193-560 與 PIASy 作用;而PIASy 則是利用 C 端胺基酸 331-510 片段與 mLRH-1 作用。已知蛋白分子可能會藉由干擾輔活化因子與轉錄因子的交互作用而達到抑制轉錄因子的效應。為了了解 PIASy 是否經由這種機制影響 mLRH-1 對CYP11A1 啟動子的轉錄作用,我們取了 LRH-1 的輔活化因子 SRC-1 與 β-catenin,以及與 PIASy 有交互作用的 TBP 蛋白作測試。結果發現只有 SRC-1 可部分回復 PIASy 對 mLRH-1 的抑制作用,因而推論 PIASy 可能會干擾 SRC-1 與 mLRH-1 的結合而影響 mLRH-1 的轉錄能力。此外,我們還發現 β-catenin 與 TBP 本身都會抑制 mLRH-1 對CYP11A1啟動子的轉錄能力。
本實驗室先前研究指出 mLRH-1 有兩段胺基酸序列 (117-168及169-193) 具有 NLS (Nuclear localization signal) 的特性,可將 GFP 綠色螢光蛋白引導至細胞核中。當我們將此兩片段分別從mLRH-1上剔除後進行免疫螢光染色分析,結果顯示刪除任何一片段,其在細胞中的分布與全長 mLRH-1 相似,只存在於細胞核中。若同時將這兩片段剔除後,mLRH-1則會均勻分散至細胞質與細胞核中。這說明 mLRH-1 具有兩個 NLS 片段,只要有一個 NLS即可使 mLRH-1 專一表現於細胞核中。 Liver receptor homolog-1 (LRH-1) is a transcription factor and belongs to nuclear receptor 5A subfamily. LRH-1 is mainly expressed in liver、intestine and in ovary. LRH-1 has been shown to enhance the tanscriptional activity of several steriodogenic promoters including CYP11A1. Our previous studies indicated that PIASy markedly inhibited the mLRH-1-stimulated activity of the human CYP11A1 promoter. This study investigates the molecular mechanism of the expression of PIASy on mLRH-1-mediated transcription. A series of fusion proteins of Gal4-mLRH-1 and VP16-PIASy were constructed. Mammalian two-hybrid assay demostrates the protein-protein interaction between mLRH-1 and PIASy. In addition, the C-terminal residues 193-560 of mLRH-1 and C-terminal of PIASy are invovled in this interaction. We futher tested whether PIASy inhibits mLRH-1 transactivity by interfering the interaction between mLRH-1 and coactivators. We found that SRC-1 can partially rescued the PIASy inhibition of mLRH-1 transactivity. Furthermore, mammalian two-hybrid assay recealed that PIASy reduced the interaction between mLRH-1 and SRC-1.It suggests that PIASy may function as a repressor of mLRH-1 by interfering the action of SRC-1. In addition, we found that both β-catenin and TBP can inhibit the mLRH-1 sitimulated CYP11A1 promoter activity. Our previous studied indicated that the DNA binding domain of mLRH-1 may contain two nuclear localization signaling peptides (117-168 and 169-193). In this study, we examined the location of mutated by immunocytochemistry. Deletion of residues 116-169 or 169-193 has no effect on nuclear localization of mLRH-1. However, removal of residues 116-193 results in a evenly distribution of mLRH-1 in nucleus and cytoplasm. It suggested that either 116-169 or 169-193 is sufficient for nuclear localization of mLRH-1. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27176 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生理學科所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-97-1.pdf Restricted Access | 1.06 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.