建立泛素化反應之高靈敏度螢光檢驗法

Abstract

泛素可經由C端甘胺酸（glycine）與目標蛋白的賴胺酸（lysine）側鏈形成共價鍵結—異胜肽鍵（isopeptide bond），這個過程稱為泛素化。此外，因為泛素分子上具有七個賴胺酸，因此其本身也可以進行泛素化，形成聚泛素鏈（polyubiquitin chain）。不同的聚泛素鏈可將目標蛋白引導至細胞內不同的位置以及反應途徑，因此在做泛素化的研究時，辨識不同的聚泛素鏈是一個很重要但困難的工作。在本篇研究中，我們想要建立一個利用螢光標定的方法區分不同種類泛素化的辨識系統。我們先建立三種在不同位點的點突變泛素，每一種都有一個半胱胺酸（cysteine）的取代或插入，於是我們便可以使用易與硫醇基反應的（thiol-reactive）螢光分子標定突變的泛素。接著我們以酵母菌的RNA聚合酶II（Pol II）做為泛素化的目標蛋白，並以螢光標定的泛素進行泛素化反應。實驗結果顯示，在三種我們所建立的螢光標定泛素中，有兩種可以結合到Pol II上，並為螢光成像（fluorescence imaging）所偵測。此外，我們也發現了一個在Pol II上未曾報導過的E3獨立泛素化反應。由以上結果得知，我們的方法可以用來偵測受泛素化修飾的蛋白，並且可以避免非專一反應所產生的不確定性；而螢光偵測的高敏感度，也提供我們一個發掘未知泛素化途徑的工具。

Ubiquitin can be covalently conjugated to other proteins through an isopeptide bond formed between the Lys side chain of the target protein and the C-terminal Gly of ubiquitin, and the process is called ubiquitination. This ubiquitin molecule could be further ubiquitinated to form polyubiquitin chain. Ubiquitin has totally seven Lys which could be used in polyubiquitination. The polyubiquitin chains linked by different Lys lead the ubiquitinated target proteins to different cell locations and pathways, hence polyubiquitin chain identification is an important but difficult work for investigating ubiquitination. In this study, we aimed to construct a fluorescence detection system for identifying ubiquitination. We constructed three ubiquitin mutants by introducing cysteine residue in three different sites, and then thiol-reactive fluorescent dye were labeled to the cysteine residue of these mutants. Using RNA polymerase II (Pol II) as ubiquitination target and these dye-labeled ubiquitin mutants to proceed ubiquitination, we have shown that 2 out 3 dye-labeled ubiquitin mutants could be conjugated to Pol II and detected by fluorescence imaging. Furthermore, we found an unreported E3 independent ubiquitination on Pol II. In conclusion, our method allows us to detect ubiquitinated proteins without the uncertainties from non-specific interactions, and the high sensitivity of this detection system could provideus a new way to discover unknown ubiquitination pathways.