甘藷IbSUMO結合蛋白之釣取與研究

Abstract

蛋白質的轉譯後修飾作用在調節蛋白質作用上扮演一個重要的角色。Small ubiquitin-related modifier (SUMO)是近年來被發現與ubiquitin蛋白結構相似，且作用機制也與ubiquitination相似。SUMO以共價類胜肽鍵(isopeptide bond)的方式與其目標蛋白(target protein)上的Lys產生鍵結，進而修飾目標蛋白質的活性、穩定性或細胞內座落的位置等，而這個過程稱為sumoylation。Sumoylation影響許多植物生長發育與逆境的調控，包含開花時間、非生物性逆境反應等。然而，sumoylation影響這些訊息傳遞分子的詳細機制目前仍然不清楚。在實驗室先前調取一氧化氮(nitric oxide, NO)誘導蛋白的研究中，以蛋白質二維電泳系統(two-dimensional electrophoresis system)分離甘藷葉片(Ipomoea batatas cv. Tainung 57)在處理NO後的差異性蛋白, 再以LC/MS/MS分析，發現IbSUMO2為NO所誘導的蛋白質之一。此外，也在阿拉伯芥中建構大量表現GFP-IbSUMO2-GFP融合蛋白系統，進而來觀測IbSUMO2的細胞位置，結果顯示IbSUMO2主要坐落在細胞核；然而有趣的是，植株在處理過氧化氫(Hydrogen peroxide, H2O2)下，位於細胞核的IbSUMO2會轉移到細胞質。因此，為了瞭解IbSUMO2在細胞間移動的機制，我們利用甘藷及阿拉伯芥系統來調取IbSUMO2結合的目標蛋白。在甘藷系統中，主要以蛋白質二維電泳分離系統來找出H2O2誘導下可被IbSUMO2修飾之蛋白，而在阿拉伯芥系統，則以免疫沉澱(immunoprecipitation, IP)進行分離IbSUMO2修飾之蛋白，最後再以LC/MS/MS鑑定分析蛋白身分。根據液相層析質譜儀分析結果，我們獲得三個可能會被IbSUMO2結合的蛋白，分別為BiP1 (Luminal-binding protein 1)、putative F-Box protein以及HSC70-1 (Heat shock cognate 70 kDa protein )，且在H2O2處理下這三個基因的表現量也會被誘導上升，推測這三個蛋白質極可能受到IbSUMO2調控來參與氧化逆境的反應。

Post-translational modifications of proteins play critical roles in regulation of protein activities. Small ubiquitin-related modifiers (SUMOs) were discovered to resemble ubiquitin in their three-dimensional structures and the way they link to other proteins. SUMO becomes covalently linked by an isopeptide bond between its C-terminal glycine and the lysines within the target proteins. Sumoylation controls a broad spectrum of cellular activities, including roles in changing protein activities, stabilizing proteins, and nuclear trafficking. Protein sumoylation plays an important role in plant development, flowering-time regulation, and abiotic stress response. However, the molecular roles of sumoylation in these pathways are largely unknown. In the previous studies in our laboratory, the leaves of sweet potato (Ipomoea batatas cv. Tainung 57) were treated with nitric oxide (NO), and the abundance of several proteins was increased based on the analyses of 2D electrophoresis. After the identification of LC/MS/MS, IbSUMO2 is one of the NO-induced proteins. Furthermore, fusion protein GFP-IbSUMO2-GFP was constructed to study the localization of IbSUMO2 within Arabidopsis cells by confocal microscopes. Results indicated that most GFP- IbSUMO2-GFP was in nucleus, and, however, after H2O2 treatment GFP-IbSUMO2-GFP translocated to cytoplasm in Arabidopsis cells. In order to understand the mechanism of IbSUMO2 translocation, a method to isolate IbSUMO2-conjugated proteins from sweet potato and Arabidopsis was developed. The effect of H2O2 on the accumulation of IbSUMO2 conjugates in sweet potato was studied by 2D electrophoresis. In addition, immunoprecipitation was used to isolate the putative proteins modified by IbSUMO2 in Arabidopsis after H2O2 treatment, and sumoylated proteins were identified by LC/MS/MS. According to the LC/MS/MS data, I found three putative proteins could be sumoylated after H2O2 treatment, they were BiP1 (Luminal-binding protein 1), putative F-Box protein, and HSC70-1 (Heat shock congnate 70 KDa protein). RT-PCR indicated that these three transcripts were also induced by H2O2, indicating that sumoylation of these three proteins played important roles during oxidative stress.