- 學位論文
蛋白酶體 19S Rpt3 ATPase 之 SUMO 化修飾研究
Study of the SUMOylation of Proteasome 19S Rpt3 ATPase
摘要
Rpt3 (regulatory particle triple-A ATPase 3) 為蛋白酶體19S regulatory particle (RP) 的次單元體之一，與其他五個不同的AAA-ATPase 次單元共同組成19S RP的基座 (base)，主要參與辨識 poly-ubiquitin chain、蛋白質unfolding、運送目標蛋白質進入蛋白酶體活性中心等。 本研究室在非洲草綠猴腎臟細胞 (COS7 cell) 中發現Rpt3 為SUMO2 (smallubiquitin-like modifier2) 的受質之一。本研究於真核宿主細胞轉染表現Rpt3，並觀察到Rpt3於人類胚胎腎臟細胞 (HEK293T cell) 中同時受SUMO1與SUMO2修飾。透過於細胞中表現不同長度Rpt3以及突變位於保守性序列Ψ-K-x-D/E當中的離胺酸K62、K69與K408，都未能鑑定出Rpt3受SUMO化的確切位置。然而，將Rpt3所具備之 SUMO-interacting motif (SIM): SIM3的疏水性胺基酸突變為alanine後，確實降低了 Rpt3 的SUMO化程度，表示SIM3可能參與在Rpt3的SUMO化機制之中，且提供了Rpt3與受SUMO1修飾蛋白質產生非共價交互作用的可能性。此外，以活細胞影像觀察GFP-Rpt3在細胞的分布情形時發現，GFP-Rpt3主要分布於細胞質內且有細胞族群可觀察到GFP-Rpt3進入到細胞核內。將細胞以蛋白酶體之抑制劑MG132處理後，觀察到GFP-Rpt3位於核內的少數細胞族群僅微幅上升。因此，關於Rpt3的SUMO化程度是否會直接的影響本身的生理功能或與其他蛋白質的交互作用等議題仍有待進一步的研究。
並列摘要
Rpt3 (regulatory particle triple-A ATPase3) is a subunit of the proteasome 19S regulatory particle (RP). The primary functions of 19S RP include recognition of ubiquitinated target proteins, unfolding and translocation of target proteins into the catalytic cavity of the 20S core particle. In our previous study, Rpt3 was found to be SUMOylated by SUMO2 in COS7 cells. In this study, the strong SUMOylation of Rpt3 by SUMO1 and SUMO2 was clearly observed in HEK293T cells. However, the exact SUMOylation site of Rpt3 was not determined by expression of truncated Rpt3 and Rpt3 mutants containing K62R, K69R and K408R mutations within the Ψ-K-x-D/E SUMOylation consensus motifs. Nonetheless, the SUMO-interacting motif SIM3 on Rpt3 may be involved in modulating Rpt3 SUMOylation, as mutation of SIM3 reduced the SUMOylation level of Rpt3 in HEK293T cells. By using the live cell imaging to analyze the expression of GFP-Rpt3 fusion protein in HEK293T cells, the data showed that GFP-Rpt3 was primarily distributed in the cytoplasm, and a small population of cells containing GFP-Rpt3 in the nucleus. However, the cell population containing nuclear GFP-Rpt3 only slightly increased after treatment of proteasome inhibitor MG132. This study suggests that Rpt3 is SUMOylated by SUMO1 and SUMO2 in HEK293T cells. However, the detailed SUMOylation mechanism and the SUMOylation sites of Rpt3 remain elusive.