蛋白酶體19S Rpt5 ATPase受SUMO化修飾之調控機制研究

Study of the regulation mechanism of the SUMOylation of proteasome 19S Rpt5 ATPase

魏綺(Chi Wei)

指導教授 ： 張世宗

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10138

摘要

26S蛋白酶體 (26S proteasome) 之19S regulatory particle具有辨識泛素鏈、對蛋白質進行去摺疊、開啟20S之基質通道入口等功能。19S regulatory particle ATPase 5 (Rpt5) 與其他五個Rpt次單元體共同組成19S的基座 (base)，並且與20S之alpha-ring結合，進而調控蛋白酶體之活性。
本實驗室先前的研究發現Rpt5在COS7細胞中受到SUMO2 (small ubiquitin-like modifier 2) 修飾，利用大腸桿菌SUMO修飾系統也證明Rpt5會被SUMO1與SUMO2修飾。此外也發現Rpt5具有類似SUMO-interacting motif (SIM) 的序列。將其中的SIM3突變後，Rpt5在大腸桿菌SUMO修飾系統中被SUMO修飾的情形顯著地減少了，因此我們推論Rpt5上之SIM應與其被SUMO化修飾有關。
本研究進一步以HEK293T細胞為材料，來探討SIM是否直接參與了Rpt5之SUMOylation。在HEK293T細胞中，能觀察到Rpt5有受SUMO1及SUMO2/3修飾的現象，但是突變SIM3後，Rpt5被SUMO修飾的程度不受影響，顯示SIM3可能不是Rpt5具有功能的SIM，亦或者有其他調控方式存在。而利用胞外去SUMO化 (deSUMOylation) 酵素反應，以SUMO1/sentrin specific peptidase 1 (SENP1) 處理在細胞中受SUMO修飾的Rpt3及Rpt5，可以發現其SUMOylation程度有所下降，顯示Rpt3及Rpt5受SUMO修飾的現象可以被SENP1辨識並進行截切。另外，將Rpt3及Rpt5進行胞外SUMO化酵素反應後，並沒有觀察到Rpt3及Rpt5有受SUMO修飾的現象。

關鍵字

Rpt5 ； SUMO ； SIM ； SUMOylation ； SENP1

並列摘要

Regulatory particle triple-A ATPase 5 (Rpt5) is one of the subunits of the 19S regulatory particle of 26S proteasome, which forms the 19S base together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. The 19S base performs several functions, such as recognizing polyubiquitin chain, unfolding substrate, gate opening of the 20S core particle, and translocation of target proteins. Our previous study has revealed that Rpt5 was modified by small ubiquitin-like modifier2 (SUMO2) in COS7 cells. In addition, Rpt5 was modified by SUMO1 and SUMO2 in the in vitro E. coli SUMOylation assay system. Furthermore, Rpt5 contains several putative SUMO interacting motifs (SIMs), and the in vitro experimental results showed that SUMOylation of Rpt5 by SUMO1 was markedly inhibited while SIM3 was mutated.
In this study, HEK293T cells were utilized to investigate the function of SIM3 in Rpt5. The results showed that Rpt5 was modified by SUMO1 and SUMO2 in HEK293T cells; however, the SUMOylation level of Rpt5 was not reduced when SIM3 was mutated, which suggesting SIM3 is probably not involved in the SUMOylation of Rpt5.
The in vitro deSUMOylation assay was conducted to examine whether the SUMO conjugates of Rpt3 and Rpt5 obtained from HEK293T cells can be removed by SUMO1/sentrin specific peptidase 1 (SENP1). The data showed that the SUMO conjugates of Rpt3 and Rpt5 were efficiently cleaved by SENP1, suggesting that the formation and de-conjugation of SUMO conjugates on Rpt3 and Rpt5 were reversible.
The in vitro SUMOylation assay showed that Rpt3 and Rpt5 from HEK293T cells were not modified by SUMO2.

並列關鍵字

Rpt5 ； SUMO ； SIM ； SUMOylation ； SENP1

參考文獻

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