2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648336摘要：泛素-蛋白酶體路徑是一重要的蛋白質降解系統，以維持細胞蛋白質的恆定。E3泛素連接酶負責 辨識蛋白質，協助將泛素鍵結至特定的蛋白質，促使蛋白質經由蛋白酶體而降解。E3泛素連接酶參與 各種細胞作用，並與多種人類疾病有所關連。RNF115是屬於具有RING結構類型的E3泛素連接酶，先 前研究發現RNF115過量表現於乳癌腫瘤，且RNF115的高度表現與乳癌細胞的增生有顯著正關聯 性。還有研究指出，RNF115與Rab7蛋白質有交互作用，會影響細胞内囊泡的運輸。除此之外，尚未 有RNF115生理作用之相關研究。Liver receptor homologue-1 (LRH-1)屬於核受器NR5A家族之成員主要表現於肝臟、膜臟、小 腸、卵巢及脂肪組織。LRH-1為一轉錄因子調控很多基因的表現，影響了各種不同的生物反應，包括 發育、代謝、類固醇荷爾蒙生成與腫瘤生成。我們先前的研究顯示，LRH-1蛋白質相當不穩定且會經 由泛素-蛋白酶體而被降解。我們進一步找到與LRH-1有交互作用的蛋白質DDB2，說明其藉由 CUL4-DDB1 E3泛素連接酶的作用，會促進LRH-1蛋白質降解。近來，我們又發現新的E3泛素連接酶 RNF115與LRH-1有交互作用。有趣的是，RNF115並不會造成LRH-1蛋白質降解；相反地，LRH-1會顯 著降低RNF115蛋白質的量。AMP活化激酶（AMPK)是調控肝臟葡萄糖與與脂肪代謝的重要分子。前人研究顯示在乳癌細胞 RNF115會抑制AMPK的活化，但其在肝臟的作用則尚未研究。由於LRH-1在肝臟葡萄糖與與脂肪代謝 也扮演重要角色，促使我們深入探討LRH-1調控RNF115蛋白質穩定性的機制。我們假設此作用在 AMPK調控肝臟代謝路徑可能具有相當程度的重要性。此外，我們還發現RNF115大量表現於小鼠的 睪丸組織，免疫染色分析顯示其專一的存在於睪丸的賽托利細胞（Sertoli cells)與長型的精細胞 (elongated spermatids)，此結果顯示RNF115對睪丸分化與精子生成可能扮演重要角色。研究RNF115 在睪丸的生理功能將有助於我們對調控精子生成機制的了解。此計晝的主要研究目標包括：目標一：研究LRH-1調控RNF115蛋白質穩定性的機制 目標二：探討RNF115在肝臟代謝的角色 目標三：探討RNF115在雄性生殖的角色<br> Abstract: Ubiquitin-proteasome pathway is an important proteolytic system to maintain the homeostasis of cellular proteins. E3 ubiquitin ligases are responsible for conjugating ubiquitins to specific protein and usually targets them for proteasomal degradation. The E3 ligases are involved in numerous cellular processes and linked to multiple human diseases. RNF115 is a RING finger domain ubiquitin E3 ligase. RNF115 was found to be overexpressed in breast cancer and its expression is significant linked to breast cancer cell proliferation. RNF115 has been shown to interact with Rab7 to affect vesicle traffic. However, the physiological functions of RNF115 are poorly understood.Liver receptor homologue-1 (LRH-1; NR5A2), a member of the orphan nuclear receptor NR5A subfamily, is mainly expressed in liver, pancreas, intestine, ovary and adipose tissue. LRH-1 functions as a transcription factor to regulate the expression of numerous genes associated with diverse biological processes, including development, metabolism, steroidogenesis and carcinogenesis. We previously found that LRH-1 is extremely unstable and is destroyed by ubiquitin-mediated proteolysis. We have identified that DDB2 is a LRH-1 interacting protein and mediates LRH-1 ubiquitination/degradation by CUL4-DDB1 E3 ligase. Recently, we found that RNF115 is a novel partner protein of LRH-1. Interestingly, RNF115 E3 ligase had no effect on LRH-1 protein degradation; on the contrary, its protein levels were negatively regulated by LRH-1.AMP-activated kinase (AMPK) functions as a critical regulator of glucose and lipid metabolism in liver. RNF115 has been shown to be an endogenous inhibitor of AMPK activation in breast cancer cells, but its function in liver is unknown. Due to the important roles of LRH-1 in glucose and lipid metabolism in liver, we therefore focus on underlying mechanism by which LRH-1 regulate RNF115 protein levels in this proposal. We speculate that LRH-1-mediated RNF115 protein stability may contribute a part to the AMPK-mediated metabolic pathway in liver. In addition, we found that RNF115 is highly expressed in mouse testis and immunostaining analysis revealed that RNF115 is specifically present in the Sertoli cells and elongated spermatids. This implies that RNF115 may play a role in the testis differentiation/spermatogenesis. Identification of the biological functions of RNF115 in testis will increase our understanding in the regulation of spermatogenesis. The specific aims of the proposal are:Aim 1: To elucidate the mechanism by which LRH-1 regulates RNF115 protein stabilityAim 2: To investigate the role of RNF115 in hepatic metabolismAim 3: To explore the physiological function of RNF115 in male reproductionRING泛素連接酶RNF115蛋白質核受器LRH-1AMP活化激酶賽托利細胞RING ubiquitin ligaseRNF115Liver receptor homologue-1 (LRH-1)AMP-activated kinaseSertoli cells