https://scholars.lib.ntu.edu.tw/handle/123456789/159918
標題: | Nedd8對cullin-RING泛素接合脢及其受質的影響 The effects of Nedd8 on cullin-RING ubiquitin ligases and their substrates |
作者: | 吳君泰 Wu, June-Tai |
關鍵字: | 泛素;cullin-RING接合脢;cullin;Nedd8;CSN複合體;neddylation;deneddylation;Ubiquitin;cullin-RING ligase (CRL);CSN complex | 公開日期: | 2006 | 摘要: | 泛素是一種小分子胜肽,泛素—蛋白解體系統負責許多細胞生理所必需的蛋白質降解.整個蛋白質降解的過程先是由泛素接合脢把泛素專一性地結合在即將被降解的標的蛋白上,再經過重複的接合脢作用,標的蛋白上聚合出長鏈型泛素聚合物,帶有長鏈型泛素聚合物的標的蛋白隨即被蛋白解體所辨識,並且被降解成短胜肽產物.細胞內有很多泛素接合脢,其中有一類稱為cullin-RING接合脢(簡稱CRL).完整的CRL是個由cullin蛋白為基石所建構的蛋白複合體.負責延攬受質的受質受器在cullin 的N端,而負責銜接泛素配合酵素(E2)的Roc1則座落在cullin的C端. 接合脢的酵素反應可視為CRL上的泛素藉由地緣之便,傳遞到同在CRL上的受質.Nedd8是胺基酸序列和三級結構都與泛素十分類似的一種小分子胜肽.它具有類似泛素的性質夠過neddylation的酵素反應能夠結合在其他蛋白如cullin之上.相反地透過CSN複合體催化的deneddylation酵素反應,Nedd8又可以從neddylated cullin上被切下來.過去的研究指出,CRL經過neddylation之後才能有效率的執行泛素接合脢的功能,可是有關deneddylation如何影響CRL酵素活性的了解甚少.本論文主要內容在於探討CSN突變果蠅的活體細胞內,cullin1和cullin3的穩定性變差.cullin穩定性變差的主要原因是在CSN突變果蠅裡,cullin一旦發生neddylation後就無法經由deneddylation回復到穩定的原態.結果細胞不再能夠保有足夠的cullin儲備,於是CRL的活性不但沒有因為neddylation而提高,反而因為cullin普遍不足而下降.在CSN突變果蠅細胞中,Nedd8和受質受器Slimb也變得特別不穩定.CSN突變和Nedd8突變,或和cullin突變,或和Slimb突變互相加成對果蠅的存活率影響,表示CSN調節CRL組成成員以及Nedd8的穩定性具有生理意義.但是對於特定的CRL受質如Ci,在CSN突變細胞中增加cullin1和Nedd8的量,不但不能藉由彌補CSN突變細胞中cullin1,Nedd8的儲備量而提高CRL的酵素活性,反而弔詭地減低了CRL的酵素活性,表示cullin1, Nedd8的量對CRL的酵素活性在不同deneddylation程度下會出現兩種截然不同的影響.論文最後探討了同樣是SCFSlimb接合脢受質的兩種蛋白質,被泛素--蛋白解體系統降解的程度受到CSN突變的影響卻有所不同,表示受質能夠左右CRL接合脢活性受neddylation/deneddylation調節的程度. Ubiquitin-proteasome mediated proteolysis is an essential pathway involved in many cellular processes. Ubiquitin is a small polypeptide. Polymerized ubiquitin chains are first attached to the proteins designated to be degraded by a substrate specific ubiquitin E3 ligase, a process called ubiquitylation. The ubiquitylated protein is then recognized by the 19S proteasome and subsequently degraded by the 20S proteasome. Cullins are scaffold proteins that assemble substrate binding receptors/adaptors, and Roc1 into cullin-RING E3 ligase (CRL) complexes transferring ubiquitin moieties onto many cellular proteins. Nedd8 is an ubiquitin-like polypeptide that can be transferred onto the conserved lysines of cullins, a process called neddylation. Deneddylation, the reverse reaction that removes the Nedd8 moieties from cullins, is carried out by COP9 signalosome (CSN). Neddylation is essential for CRL activities in vivo. How deneddylation affects the activities CRLs hasn’t been fully explored. In this thesis, I first showed that cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable in CSN mutant cells. Moreover, deneddylating activity in CSN is required to preserve Cul1 and Cul3 protein levels. In addition to cullins, Nedd8 and Slimb, one of the substrate receptors in Cul1 organized E3 (SCF), are also unstable in CSN mutant cells. These data indicate that the several CRL components could be protected by CSN from neddylation-induced degradations. The significance of CSN-mediated protection on CRLs is suggested by synergistic interaction between CSN5 mutant and several cullin mutants, Nedd8 mutant, as well as a dominant-negative Slimb mutant. Because neddylation-induced degradation of Slimb depends on SCF activity, rescuing Cul1 and Nedd8 protein levels in CSN mutant cells was found to paradoxically compromise SCFSlimb activity. This result is possibly due to the enhanced neddylation-induced degradation of Slimb. The significance of neddylation-induced degradation of cullins and Nedd8 is to preserve substrate degradation in this context, implying another layer of complexity in regulating CRL activity through neddylation and deneddylation. Finally, I found the protein levels of two SCFSlimb substrates changed differently in CSN mutant cells, implying that substrates may play a role on modulating the effects of neddylation and deneddylation on SCFSlimb |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/51389 | 其他識別: | en-US |
顯示於: | 分子醫學研究所 |
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ntu-95-D92448003-1.pdf | 23.31 kB | Adobe PDF | 檢視/開啟 |
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