Title

阿拉伯芥關鍵轉錄因子EIN3蛋白質之結構與功能性分析

Translated Titles

Structural and functional analysis of the key transcription factor ETHYLENE INSENSITIVE3 in Arabidopsis thaliana

Authors

李賢啟

Key Words

乙烯 ; 轉錄因子 ; EIN3 ; Ethylene ; transcription factor ; ETHYLENE INSENSITIVE3 ; EIN3

PublicationName

中興大學生命科學系所學位論文

Volume or Term/Year and Month of Publication

2018年

Academic Degree Category

碩士

Advisor

王隆祺

Content Language

繁體中文

Chinese Abstract

乙烯是一種調節植物生長、發育以及各種逆境反應的氣態植物賀爾蒙,主要調控花瓣凋謝、葉片老化以及果實成熟等。當乙烯與受體結合時,CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) 失去活性而無法進行ETHYLENE INSENSITIVE2 (EIN2) 的磷酸化,EIN2的C端 (C terminus) 則藉著蛋白水解作用而轉移到細胞核中使EIN3和EIL1 (EIN3-LIKE1) 蛋白結構穩定, EIN3 / EIL1所扮演的角色是在阿拉伯芥中調控眾多乙烯反應基因表達的關鍵轉錄蛋白質,而EIN3蛋白的二聚化是影響其轉錄活性的重要因素。 先前的研究揭示了EIN3的核心DNA-binding domain (DBD) (174-306 a.a.) 包含兩個鹼性結構域,BD-III (238-248 a.a.) 、BD-IV (265-274 a.a.) 以及一個富含脯氨酸的區域。BD-III及BD-IV可能直接參與了蛋白質-DNA交互作用 (protein-DNA interaction,PDI) ,而富含脯胺酸的區域則可能參與蛋白質之間的交互作用 (protein-protein interaction,PPI) 。值得注意的是,在EIN3的DBD已經發現了幾個破壞EIN3功能的突變,包括EIN3P216S,EIN3K245N,及EIN3T174A,但其對於PPI及PDI的影響尚未被詳細的檢驗。此外,我們實驗室針對破壞EIN3二聚化篩選得到三種小分子化合物,分別命名為7922,2133,及2397。以分子對接分析預測,與此三個化合物形成共價鍵的胺基酸可能會影響EIN3二聚化的功能。我使用定點突變的方式產生P216、K245、T174及其鄰近胺基酸的置換突變,同時也包括與小分子化合物可能形成鍵結的胺基酸,接著利用酵母菌單,雙雜交系統分析EIN3蛋白質PDI與PPI的功能,以及使用短暫基因表達系統檢測報導基因活性與建構阿拉伯芥 (Arabidopsis thaliana) 轉殖株進行功能互補的實驗,分析EIN3蛋白質在植物細胞與個體的活性。 藉由特定區域的蛋白結構與小分子化合物分子對接分析結果所設計的胺基酸突變及後續鑑定,我找到幾個參與EIN3活性功能的胺基酸,導致突變後的EIN3 失去PPI或/及PDI的功能,基因轉錄活性的降低與轉殖株功能互補能力的喪失,此類胺基酸包括W214、W215、P216、G218、Q179、E180、L181、K245、Q294、E295。比較特別的發現是K245的突變雖然破壞了PDI的功能,但在大量表達的轉殖株中,此突變仍保有部分EIN3的活性。此外,本研究新的發現為G218也是具有影響EIN3功能之重要胺基酸。 總結此論文研究的發現,除了文獻中已知重要的EIN3胺基酸之外,其周圍的結構也同樣具有參與調控蛋白質的功能。本研究成果除了詳細解析EIN3蛋白質結構功能,提供更多針對EIN3轉錄活性的藥物研發方向,有助於進一步應用在蔬果花卉採收後熟處理以及保鮮期之調節,論文中所建立的小分子化合物篩選平台及研究方法同樣也適用於其它農作物中重要轉錄蛋白質的功能性分析。

English Abstract

Ethylene is a gaseous plant hormone regulating plant growth, development and stress responses including promotion of flower withering, leaf aging and fruit ripening. When ethylene binds to the receptors, the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) kinase is inactivated and fails to phosphorylate ETHYLENE INSENSITIVE2 (EIN2). Subsequently, the cytosolic C terminus of EIN2 (EIN2Cend) is released by proteolysis and translocated to nucleus where EIN2Cend activates EIN3 and EIL1 (EIN3-LIKE1) by suppressing protein degradation of EIN3/EIL1 that is mediated by the 26S proteasome. EIN3/EIL1 are the key transcription factors to regulate the expression of numerous genes responsive to ethylene in Arabidopsis thaliana. It has been shown that dimerization of EIN3 is important for its transcriptional activity. Previous studies uncovered the core DBD (DNA-binding domain) (174-306 a.a.) of EIN3 consisting of two basic domains (BD), BD-III (238-248 a.a) and BD-IV (265-274 a.a.), and a proline-rich region. The BD-III and BD-IV have a proposed role in mediating protein-DNA interaction (PDI) and the proline-rich region is likely involved in protein-protein interaction (PPI). Interestingly, several mutations disrupting EIN3 function including EIN3P216S, EIN3K245N and EIN3T174A are located within the core DBD of EIN3. However, their roles in PPI and PDI have not been fully examined. In addition, three small molecule compounds denoted by 7922, 2133, 2397 that were identified by chemical screens to disrupt EIN3 dimerization in our laboratory. By molecular docking analysis, I revealed a group of amino acids forming non-covalent hydrogen bonds with the compounds within the EIN3 DBD, which suggests the PPI and/PDI of EIN3 may be affected by the small molecules. To examine the functional relationship of the key residues in EIN3 DBD, I generated substitution mutations of P216, K245, and T174 and their spatially adjacent amino acids based on crystallographic studies. Similarly, substitution mutations were also introduced to the amino acid residues predicted to interact with small molecule compounds. Functional characterization was implemented to examine PDI and PPI of EIN3 by yeast one- and two-hybrid systems, respectively, to analyze transcriptional activity of EIN3 by transient gene expression assays in plant cells and to determine EIN3 functionality by complementation experiments using transgenic Arabidopsis plants. I found several amino acid residues crucially involved in EIN3 function, including W214, W215, P216, G218, Q179, E180, L181, K245, Q294, and E295. For example, EIN3K245A allele disrupts the PDI of EIN3 by Y1H assay but still retains partial function when over-expressed in transgenic plants. One of the new findings in this study is that G218 interacts with compound 2133 and is important for both PPI and PDI of EIN3 that EIN3G218 fails to complement Arabidopsis ein3-1/eil1-1 mutant, which highlights the molecular mechanism of small molecules in affecting EIN3 function. In summary, my research uncovers the mechanistic roles of important residues in EIN3 function, which include not only some of the previously known amino acids, but also those involved in interaction with small molecules disrupting PPI and PDI of EIN3. Studies on structure and activity relationship of EIN3 may provide critical information for further development of effective drugs to downregulate EIN3 function, which has practical applications on post-harvest management for fruit ripening, vegetable storage and shelf life of cut flowers. Furthermore, the methodology to screen and characterize small molecule compounds in this study can be applied to functional studies of other biologically important transcriptional factors in crop plants.

Topic Category 生命科學院 > 生命科學系所
生物農學 > 生物科學
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