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標題: | LRH-1調控RNF138蛋白質穩定性 Liver receptor homolog-1 regulates RING finger protein 138 protein stability |
作者: | Jou-Yin Meng 孟柔吟 |
指導教授: | 胡孟君 |
關鍵字: | LRH-1,RNF138,泛素-蛋白?體路徑, LRH-1,RNF138,Ubiquitin-proteasome pathway, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | Liver receptor homolog-1 (LRH-1;NR5A2) 隸屬於孤兒核受器,主要表現在肝臟、腸道、胰臟以及卵巢。LRH-1對於細胞增生、發育、膽固醇運輸、膽酸恆定以及固醇類荷爾蒙的生成等扮演重要的角色。LRH-1為轉錄因子會和輔因子產生交互作用,進而調控下游基因的表現。本論文發現LRH-1會調控E3連接酶RNF138 (RING finger protein 138) 蛋白質量,並與RNF138有交互作用。當RNF138與LRH-1同時轉染於HEK293T中,會使RNF138蛋白質的量降低,並且具有劑量依賴性。利用蛋白質衰減測定,發現LRH-1可降低RNF138蛋白質的穩定性;若給予蛋白酶體抑制劑MG132,會部分抑制LRH-1對RNF138蛋白質的影響。這些結果顯示,LRH-1可能會促進RNF138經由蛋白酶體路徑而降解。
GST pull down的分析顯示,LRH-1以DNA binding domain (DBD) 與RFN138的N端進行交互作用。有研究指出,核受器的DBD可能具有潛在的RING finger結構。我們以點突變破壞LRH-1位於DBD的潛在環狀結構 (loop),這些突變在失去轉錄活性的情況下,仍能使RNF138蛋白質量下降,說明LRH-1的DBD可能不具有E3連接酶活性,且調控RNF138蛋白質量的作用與其轉錄活性無關。位了確認LRH-1調控RNF138蛋白質量的重要區段,建構出不同的LRH-1片段,發現單獨的LBD片段能降低RNF138蛋白質的量。綜合結果顯示,LRH-1會降低RNF138蛋白質的穩定性,而此作用與其轉錄活性無關,反而是LBD在LRH-1的功能中扮演重要角色。 Liver receptor homolog-1 (LRH-1, NR5A2) is an orphan nuclear receptor, is predominantly expressed in the liver, intestine, pancrease and ovaries. LRH-1 regulates the expression of genes involved in development, metabolism, steroidogenesis and cancinogenesis. We found that LRH-1 reduced the protein level of RNF138 (RING finger protein), and they had interaction in vitro. By protein turnover assay, we found that LRH-1 decreased RNF138 protein stability. In addition, LRH-1 mediated RNF138 protien levels were inhibited by the proteasome inhibitor MG132. These results indicates that LRH-1 may promote RNF138 degradation through proteasome pathway. The GST pull-down experiments showed that LRH-1 interacted with RNF138 N-terminus by DNA binding domain (DBD). It has been reported that the DBD of nuclear receptors has potential RING finger structure. We destroyed the potential loop structure in DBD of LRH-1 by site mutagenesis. These mutants lose the transcriptional activity, but still could reduce RNF138 protein level. It suggested that LRH-1 DBD may not have E3 ligase activity and LRH-1-mediated RNF138 protein reduction was independent of its transactivity. To identify the region responsible for LRH-1 function, a series of LRH-1 truncation constructs were generated. We found that the ligand binding domain (LBD) alone could reduce RNF138 protein level. Together, these results suggested that LRH-1 can reduce RNF138 protein stability that is independent of LRH-1 transcriptional activity and LBD plays an important role in LRH-1-mediated function. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54020 |
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