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標題: | UBE3C透過VPS34的降解進而調控細胞自噬和蛋白質恆定 UBE3C-Mediated VPS34 Degradation Regulates Autophagy and Proteostasis |
作者: | Tzu-Yu Huang 黃資彧 |
指導教授: | 陳瑞華(Ruey-Hwa Chen) |
關鍵字: | UBE3C,VPS34,泛素化,細胞自噬,蛋白質毒性壓力, UBE3C,VPS34,ubiquitination,autophagy,proteotoxic stress, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 細胞自噬在細胞中是一透過溶酶體來降解的路徑,得以幫助維持細胞恆定以及適應各種壓力。VPS34 是第三類磷脂肌醇-3-激酶複合體中的一個次單元,並且產生磷脂肌醇-3-磷酸,有助於細胞自噬體的生合成。雖然已知大部分參與細胞自噬機制中的關鍵成員會走向透過泛素化降解的路徑,但是關於VPS34的泛素化和穩定性的機制卻鮮為人知。在我們實驗室過去的研究中發現一去泛素酶—TRABID會對VPS34執行去泛素化作用,進而避免VPS34走向蛋白體酶體的降解途徑來促進細胞自噬,這促使我們去尋找哪個E3連接酶可以在VPS34上接上泛素鏈。在本篇論文中,我們發現E3連接酶—UBE3C可以在活體內及試管內和VPS34結合。重要的是,減弱UBE3C會增加VPS34的蛋白質含量並且防止他走向蛋白體酶體的降解途徑。再者,在正常和飢餓條件下,減弱UBE3C會促進細胞自噬體的生合成,且細胞自噬體聚集以及脂質化LC3的增加在有無巴佛洛霉素A1處理過後的細胞中也能顯而易見。更重要的是,減弱UBE3C造成VPS34蛋白質量、細胞自噬體數量和LC3脂質化上升的現象會在減弱TRABID的情況下逆轉,說明了UBE3C和TRABID能夠透過互相拮抗調控VPS34來共同維持細胞自噬的平衡。在蛋白質毒性的壓力而非粒線體受損或飢餓的條件下,VPS34和UBE3C的結合力下降進而穩定VPS34,我們發現這些壓力條件會降低UBE3C而非TRABID被帶到吞噬泡,且我們並沒有在飢餓條件下觀察到這樣的現象。更進一步的發現在這些壓力條件下,UBE3C和蛋白質體的結合力有上升的趨勢,以及在UBE3C的N端的某個片段可能參與改變UBE3C的胞內位移。綜觀上述,我們可以得知在正常和飢餓條件下,UBE3C藉由對VPS34執行泛素化作用使其降解來抑制細胞自噬,而在蛋白質毒性壓力下,UBE3C和蛋白質體的結合力上升因而降低了他與吞噬泡的結合力,並減少與VPS34的結合力。 Autophagy is a lysosomal degradation pathway and plays a critical role in maintaining cellular homeostasis and adaption to various stresses. VPS34, the catalytic subunit of class III phosphatidylinositol 3-kinase (PI3K) complexes, is crucial for autophagosome biogenesis. Previous study in our laboratory found that the deubiquitinase TRABID reduced the polyubiquitiantion of VPS34, thereby preventing it from proteasomal degradation to promote autophagy. This prompted us to explore the E3 ligase responsible for the assembly of ubiquitin chains on VPS34. In this thesis, we show that the E3 ubiquitin ligase UBE3C interacted with VPS34 in vivo and in vitro. More importantly, UBE3C knockdown increased VPS34 abundance and prevented its proteasomal degradation. Consequently, UBE3C knockdown promoted autophagosome biogenesis in both basal and starved conditions, as evident by the accumulation of autophagosome and increased the level of lipidated LC3 in cells treated with or without bafilomycin A1. Importantly, TRABID knockdown rescued the effects of UBE3C knockdown on the upregulation of VPS34 abundance, autophagosome number and LC3 lipidation, suggesting that UBE3C and TRABID coordinately control a balanced autophagy activity via counter-regulation of VPS34. Under proteotoxic stress, but not mitochondria damage or starvation, VPS34 interaction with UBE3C was diminished, resulting in VPS34 stabilization. We showed that these stresses impaired the recruitment of UBE3C, but not TRABID, to phagophore and this regulation was not observed under starvation. Furthermore, UBE3C binding to proteasome was increased under these circumstances and a N-terminal segment of UBE3C may be involved in this change of UBE3C subcellular localization. In sum, these data indicate that UBE3C inhibits autophagy by increasing VPS34 ubiquitination and degradation in basal and starvation conditions. Upon proteotoxic stresses, the increased UBE3C binding to proteasome impairs its phagophore recruitment, thereby decreasing UBE3C binding to VPS34. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73017 |
DOI: | 10.6342/NTU201901558 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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