蛋白酶體19S Rpt5 ATPase之SUMO交互作用模組功能研究

Study of the Function of the SUMO-Interacting Motif in 19S Proteasomal Rpt5 ATPase

高翊軒(Yi-Hsuan Kao)

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(2012年)

https://doi.org/10.6342/NTU.2012.02827

摘要

Rpt5 (19S regulatory particle triple-A ATPase 5) 是組成蛋白酶體 (26S proteasome) 的19S regulatory particle中六個 AAA ATPase的其中一個次單元體。Rpt5具有許多功能，包括對目標蛋白質進行unfolding、運送目標蛋白質進入蛋白酶體的活性中心，並藉此調控蛋白酶體的功能；另外，Rpt5也能夠以一個非蛋白酶體次單元體的角色參與在其他的反應當中，較常見的是與其他蛋白質組成複合體並調控某些轉錄的過程。
本實驗室先前的研究發現，在大腸桿菌的SUMO (small ubiquitin-like modifier) 化分析系統中，即使突變了Rpt5分子上之MKSE這個SUMO化修飾保守序列的離胺酸，Rpt5仍會受到SUMO1的修飾。
本研究經過胺基酸序列分析後發現，Rpt5上具有六個可能為SUMO interacting motif (SIM) 的序列，因此推論Rpt5或許能夠與SUMO進行非共價交互作用。我們假設SIM在Rpt5受到SUMO化修飾的過程中可能扮演著重要的角色，所以將SIM序列上的疏水性胺基酸皆突變成丙胺酸 (alanine)，並利用這些Rpt5的突變株作為受質，以大腸桿菌的SUMO化分析系統來檢測，發現當SIM3、SIM4、SIM5分別突變後，Rpt5受到SUMO1修飾的情形明顯降低；SIM3突變後同樣也使得Rpt5受到SUMO2修飾的情形明顯降低。另外，利用anti-HA binding agarose pull-down assay也發現Rpt5能夠與SUMO1進行非共價交互作用，但是其突變株與SUMO1非共價交互作用的情形，與Rpt5被SUMO1所修飾的結果並不一致；此外，本研究並無觀察到Rpt5與SUMO2之間具有非共價交互作用。

關鍵字

Rpt5 ； SUMO ； SIM ； non-covalent interaction

並列摘要

Regulatory particle triple-A ATPase 5 (Rpt5) is one of six AAA ATPases of 19S regulatory particle of 26S proteasome. Rpt5 performs several functions, such as unfolding target proteins and translocating these substrates to the 20S core particle, in the regulation of the proteasome catalytic activity. Furthermore, Rpt5 can also function independently from proteasome and usually participate in part of a protein complex which involved in transcription regulation.
In the previous study, we found that Rpt5 was modified by small ubiquitin-like modifier (SUMO) in an in vitro E. coli SUMOylation assay system even though its SUMOylation consensus motif MKSE was mutated.
In the present study, we found that Rpt5 contains six putative SUMO interacting motifs (SIMs), herein named SIM1-6, suggesting that Rpt5 may interact with SUMO protein in a non-covalent way. Therefore, we hypothesized that the SIMs of Rpt5 may play an important role in its SUMOylation process. By using Rpt5 mutants, whose hydrophobic residues of the putative SIMs were substituted to alanine residues, as the substrates, we demonstrated that SUMOylation of Rpt5 by SUMO1 was markedly inhibited while SIM3, SIM4, or SIM5 was mutated, respectively. In addition, SUMOylation of Rpt5 by SUMO2 was also inhibited while SIM3 was mutated. Furthermore, the non-covalent interaction between Rpt5 and SUMO1 was observed by using anti-HA binding agarose pull-down assay. Mutation of SIM3 in Rpt5 also abolished its non-covalent interaction with SUMO1. However, the SIM4, SIM5, or SIM6 Rpt5 mutant displayed greater interaction with SUMO1. These findings suggest that different SIMs of Rpt5 may play various roles in regulation of its SUMOylation and non-covalent interaction with SUMO1. The non-covalent interaction between Rpt5 and SUMO2 was not detected in this study.

並列關鍵字

Rpt5 ； SUMO ； SIM ； non-covalent interaction

參考文獻

劉昀瑄 (2011) 蛋白酶體19S Rpt5 ATPase受SUMO化修飾之研究。碩士論文，國立台灣大學生命科學院生化科技學系碩士班。

劉邦宇 (2009) 哺乳類動物細胞中SUMO結合受質之蛋白質體學研究。碩士論文，國立台灣大學生命科學院微生物與生化學研究所。

Baba D, Maita N, Jee JG, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M (2005) Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature 435: 979-982

Baba D, Maita N, Jee JG, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M (2006) Crystal structure of SUMO-3-modified thymine-DNA glycosylase. J Mol Biol 359: 137-147

Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345-356