稻米蛋白水解酶6泛素識別區域之結構與生物物理特性研究

Abstract

氣候變化帶來了許多自然災害，例如洪水和乾旱，已經威脅到植物的生存和農作物的生產。為了抵禦洪水，植物體內有一群屬於第七群乙烯反應因子 (Group VII ethylene response factors, ERF-VIIs) 的蛋白質，可以幫助植物對抗淹水逆境。在淹水（缺氧）期間，ERF-VIIs可以促進植物生長，使植物到達水面進行氧氣交換（逃逸策略），也可以停止生長以保留能量直到洪水退去（靜止策略）。ERF-VIIs N端特異性保守序列的氨基酸MCGG為N端降解子（N-degrons），在有氧環境下會被N端規則途徑( N-end rule pathway )的酵素識別並修飾成Arg-CysO2-Gly-Gly。它最終將被蛋白水解酶6 (Proteolysis 6, PRT6) 的泛素識別區域 (Ubiquitin-recognin domain, UBR domain)識別並降解，ERF-VIIs的Cys需要被氧化，才能讓植物體內知道自己處於常氧狀態，是在植物體內特有的反應。先前研究已知在酵母菌及人類的UBR domain對不同的N-degrons的辨認性的差異。根據序列比對，我們發現植物UBR domain和其他物種的UBR domain在精氨酸R15及R17等序列有高度的保守性，但還不清楚UBR domain是如何辨識其CysO2受質專一性。綜合上述，我們想要探討植物的PRT6 UBR domain是否對CysO2有專一性，及驗證當中不同N-degrons之特異性是否與其他物種有不同之處，因此純化蛋白進行結晶，也用等溫滴定量熱法 (Isothermal titration calorimetry, iTC) 和表面電漿共振（Surface plasmon resonance, SPR）的實驗方式來揭示CysO2受質的辨認機制。最後，希望可以根據我們的研究設計PRT6 UBR domain結構域的抑製劑，防止植物ERF-VIIs在淹水前被N-end rule pathway降解，以增加植物的存活率及農作物產量。

The recent climatic changes bring a lot of nature hazards such as floods and droughts that have threatened the survival of plants and the production of agricultural crops. To resist flood, plants possess a group of proteins belonging to the Group VII ethylene response factors (ERF-VIIs) to activate downstream responses. During submergence (hypoxia), the ERF-VIIs either enhance the growth so plant can reach water surface for oxygen exchange (escape strategy) or stop the growth to preserve all energy until water recession (quiescence strategy). To prevent flood responses by ERF-VIIs during normoxia condition, ERF-VIIs are degraded by N-end rule pathway of targeted proteolysis in which oxidization plays a critical role. The amino acids MCGG (N-degrons) of N-terminal specific conserved sequence of ERF-VIIs is recognized and modified by the N-end rule pathway-specific enzymes into Arg-CysO2-Gly-Gly. It will finally be recognized and degraded by UBR domain of proteolytic enzyme 6 (PROTEOLYSIS 6, PRT6). N-end rule is highly conserved in eukaryotes and previous structural studies have revealed the substrate specificity mechanism of human and yeast UBR domain. According to structure comparison and sequence alignment, we found that the plant PRT6 UBR domain contains two highly conserved arginine residues at position 15 and 17 and a special loop that may be involved in oxidizing Cys recognition.