蛋白酶體19S Rpt5 ATPase受SUMO修飾後之活性影響研究

Study of the Proteasome 19S Rpt5 ATPase Activity Affected by SUMO Modification

黃珮慈(Pei-Tzu Huang)

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(2014年)

https://doi.org/10.6342/NTU.2014.11278

摘要

蛋白酶體 (proteasome) 由20S core particle (CP) 和19S regulatory particle (RP)所組成。而19S regulatory particle的基座(base)由六個Rpt次單元體Rpt1~6共同組成。Rpt5 (19S regulatory particle ATPase 5) 屬於AAA-ATPase family，具有辨識泛素鏈、對蛋白質進行去摺疊、以及運送目標蛋白質等功能。本實驗室先前的研究發現Rpt5在COS7細胞株中會受到SUMO2的修飾，而且也可以利用大腸桿菌系統將Rpt5進行SUMO1跟SUMO2的修飾。本研究更進一步以 in vitro sumoylation assay證明，在試管中Rpt5重組蛋白可以被SUMO1修飾。
在蛋白酶體降解蛋白質的過程中，ATP的結合與水解扮演重要的角色，因此本研究想要探討SUMO修飾是否會影響Rpt5的ATPase活性。實驗結果顯示，在受到SUMO修飾之後，Rpt5的ATPase活性會下降。此外，Rpt5上有六個可能為SUMO-interacting motif (SIM) 的序列。先前的研究指出將Rpt5的SIM3突變之後，會使Rpt5在大腸桿菌系統中被SUMO1修飾的情形顯著下降；但在HEK293T細胞中重複此實驗時，卻發現Rpt5的SUMO修飾並未下降。為了排除HEK293T內生性Rpt5的干擾，本研究嘗試使用shRNA來knockdown 內生性Rpt5的表現。但是實驗結果顯示Rpt5 knockdown的細胞株無法存活，因此仍無法充分證實SIM3是否參與Rpt5之SUMO修飾作用。

關鍵字

Proteasome ； Rpt5 ； SUMO ； sumoylation ； SIM

並列摘要

The 26S proteasome is composed of 20S core particle (CP) capped with 19S regulatory particle (RP). Regulatory particle triple-A ATPase 5 (Rpt5) is one of the subunits of the 19S RP, which forms the base of 19S RP together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. The 19S base performs several functions, such as polyubiquitin chain recognition, substrate unfolding, gate opening and also translocation of target proteins into 20S CP. Our previous study has revealed that Rpt5 was modified by small ubiquitin-like modifier 2 (SUMO2) in COS7 cells, and modified by SUMO1 and SUMO2 in the E. coli sumoylation system. In the present study, the in vitro sumoylation assay further demonstrated that recombinant Rpt5 can be modified by SUMO1.
Because ATP binding and hydrolysis play critical roles in the regulation of proteasome function, this study was aimed to examine whether SUMO modification may affect the ATPase activity of Rpt5. The result showed that the ATPase activity of Rpt5 was reduced by SUMO2 modification. Furthermore, Rpt5 was found to contain several putative SUMO interacting motifs (SIMs). Although previous in vitro experimental results showed that sumoylation of Rpt5 by SUMO1 was markedly reduced while SIM3 was mutated, the level of Rpt5 sumoylation was not lowered when SIM3 was mutated in HEK293T cells. To rule out the possibility that endogenous Rpt5 might interfere with the observation of sumoylation pattern, shRNAs were applied to knockdown the expression level of endogenous Rpt5. However, the cells with inhibited Rpt5 expression were not viable. Therefore, whether SIM3 is involved in Rpt5 sumoylation remain elusive.

並列關鍵字

Proteasome ； Rpt5 ； SUMO ； sumoylation ； SIM

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