LRH-1調控RNF115蛋白質穩定性

Abstract

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.