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標題: | LRH-1調控RNF115蛋白質穩定性 Liver receptor homolog-1 regulates the stability of RING finger protein 115 |
作者: | Wen-Ting Tseng 曾文婷 |
指導教授: | 胡孟君 |
關鍵字: | LRH-1,RNF115,BCA2, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | Liver receptor homolog-1(LRH-1, NR5A2)為一種孤兒核受器,主要表現於肝臟、小腸以及卵巢中。另外,LRH-1也是一個轉錄因子,調控許多和細胞生長、代謝以及固醇類荷爾蒙生成有關的基因。除此之外,LRH-1也參與癌細胞增生轉移的調控。RING finger protein 115(RNF115)為一種E3泛素連接酶,藉由其結構上的RING區段將目標物質接上泛素,進行泛素化作用。作為RING type的E3連接酶,它也具有將自身泛素化而降解的調控能力。
在本論文中,透過GST pull sown的實驗我們發現,RNF115與LRH-1上的DNA biding domain ( DBD ) 以及Ligand binding domain ( LBD ) 具有交互作用。當RNF115和LRH-1同時轉染於HEK293T細胞中,LRH-1蛋白質量並沒有降低的情形;相反的,RNF115卻顯著降低。隨著LRH-1劑量增加,RNF115被抑制的程度亦隨之增加。另外,利用蛋白質衰減測定,我們發現LRH-1會降低RNF115蛋白質的穩定性。若在我們給予MG132處理後,LRH-1對RNF115蛋白質量的減少作用有顯著性的減緩。此結果說明,蛋白酶體系統降解路徑對於LRH-1調控RNF115扮演相當重要的角色。先前的文獻指出,核受器的DBD區域可能潛在有RING finger的結構。將LRH-1 DBD片段剔除後,對RNF115作用顯著降低。另外將位於LRH-1 DBD鋅指結構上幾個重要位點進行突變後,LRH-1失去轉錄活性,而RNF115蛋白質量的減少大幅減緩。除此之外,LRH-1中LBD片段剔除以及將LBD上AF-2區域與輔助因子連接的重要位點突變後,RNF115的量顯著恢復。綜合結果顯示,LRH-1主要經由泛素-蛋白酶體路徑促進RNF115的降解,而LRH-1中的DBD與LBD在此調控中可能扮演著相當重要的角色。 Liver receptor homolog-1 (LRH-1, NR5A2), an orphan nuclear receptor, is mainly expressed in liver, intestine and ovary. LRH-1 is a transcription factor and regulates the expression of genes involved in development, metabolism and steroidogenesis. In addition, LRH-1 is implicated in several cancers. RING finger protein 115(RNF115)is an E3 ubiquitin ligase that contains a RING domain to help the conjugation of ubiquitin to the substrate proteins including itself. In this study, we performed GST-pull down experiments to show that LRH-1 interacted with RNF115 through DNA binding domain(DBD)and ligand binding domain (LBD). When co-transfected with RNF115 in HEK293T cells, the level of LRH-1 protein was not altered. In contrast, we found that the protein level of RNF115 dramatically reduced in the presence of LRH-1. The cycloheximide-based pulse-chase assays revealed that LRH-1 reduced the protein stability of RNF115. Treatment with proteasome inhibitor MG132, but not lysosomal inhibitor, significantly attenuated the inhibitory effects of LRH-1 on RNF115 protein levels. The data suggested that LRH-1 promotes the degradation of RNF115through proteasome system. Recent studies indicated that the DBD of nuclear receptor PPARγ has the potential to form RING finger-like structure and possess the E3 ubiquitin ligase activity. We found that truncated LRH-1with DBD deletion attenuated the inhibitory effects of LRH-1 on RNF115 protein levels. Experiment with LRH-1 mutants showed that the zinc finger region in DBD, but not the transcriptional activity, was important for LRH-1-induced RNF115 degradation. The AF-2 in LBD is an important region for the interaction between LRH-1 and coregulators. LBD deletion or AF-2 mutation decreased LRH-1-induced RNF115 degradation.Our results suggested LRH-1 negatively regulates RNF115 protein stability through ubiquitin proteasome degradation pathway and the DBD and LBD of LRH-1 may play some roles in this process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7867 |
DOI: | 10.6342/NTU201603267 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2026-12-31 |
顯示於系所單位: | 生理學科所 |
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ntu-105-1.pdf 此日期後於網路公開 2026-12-31 | 2.46 MB | Adobe PDF |
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