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標題: | 阿拉伯芥組蛋白去乙醯化酶HDA6之結構及其酵素活性分析 Structural and Enzymatic Activity Studies of Arabidopsis Histone Deacetylase 6 |
作者: | Wen-Jiun Wang 王雯君 |
指導教授: | 鄭貽生(Yi-Sheng Cheng) |
關鍵字: | 阿拉伯芥,組蛋白去乙醯化酵素,三維結構重組, histone deacetylases,EMAN,3D reconstruction, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 真核生物藉由組蛋白的修飾作用,來改變染色質的組成結構,進而調控基因的轉錄活性,在表觀遺傳學 (Epigenetics) 領域中扮演著十分重要的角色。組蛋白的乙醯基化修飾通常會活化基因轉錄機制,相反地,組蛋白的去乙醯化修飾則會抑制基因表現程度。組蛋白乙醯基化程度是由組蛋白乙醯基轉移酶 (Histone acetyltransferase, HAT) 與組蛋白去乙醯化酶 (Histone deacetylase, HDAC) 這兩種互相拮抗的酵素所調控。阿拉伯芥組蛋白去乙醯化酶HDA6,屬於組蛋白去乙醯化酶中的RPD3/HDA1家族,目前已知其參與植物生長發育階段與環境逆境下的基因調控。然而,關於阿拉伯芥組蛋白去乙醯化酶的蛋白質結構卻鮮少被討論,在本研究中,我們利用穿透式電子顯微鏡技術觀測阿拉伯芥HDA6蛋白質的外觀型態,結果顯示HDA6會彼此聚集形成直徑約25奈米的橢圓形超級結構。此外,在組蛋白去乙醯化酶的活性分析上,我們將阿拉伯芥HDA6分別大量表現於大腸桿菌或阿拉伯芥中,不同於活性低落的HDA6重組蛋白,由HDA6大量表現的轉殖株中純化所得的內生性HDA6蛋白質,能測得顯著的組蛋白去乙醯化酶活性。我們進一步利用質譜技術來探究由這兩種表現系統所純化出的HDA6蛋白,在組蛋白去乙醯化酶的活性表現差異,是否有後轉譯修飾參與其中?
本論文藉由穿透式電子顯微鏡對阿拉伯芥HDA6超級結構進行電顯取象,重建出解析度約為15.3A之三維蛋白質結構模型;接下來,我們利用質譜技術首次證實阿拉伯芥HDA6與人類第一亞類HDACs類似,亦受到後轉譯修飾的調控,成功鑑定出阿拉伯芥HDA6在細胞中受磷酸化修飾的兩個胺基酸位點 (Ser427及Ser429),這兩個磷酸化位點在第一亞類HDACs中具有序列保守性,因此可能扮演了影響阿拉伯芥HDA6組蛋白去乙醯化活性的重要角色。 In eukaryotes, histone modifications regulate many developmental processes by controlling dynamic changes in chromatin structure for gene expression. Histone acetylation is often correlated with transcriptional activation, whereas histone deacetylation is involved in transcriptional repression. Histone acetylation homeostasis is determined by antagonist actions of histone acetyltransferases and histone deacetylases. The Arabidopsis thaliana histone deacetylase 6 (AtHDA6) has diverse roles to regulate genome stability, development status and environmental stress responses in plants. The molecular structures of the histone deacetylases in Arabidopsis have not been resolved for revealing the regulation in molecular level. In this study, we reported a ~25 nm spherical structure of HDA6 superstructure using transmission electron microscopy. For functional assays, we identified different expression patterns of HDAC activity between the endogenous and recombinant HDA6 proteins. In contrast of barely detectable HDAC activity in recombinant HDA6, the HDA6 extracted from 35S: GFP-HDA6 plants showed obviously HDAC activity. Here we presented the 3D structural reconstruction model of HDA6 superstructure, the resolution was about 15.3 A. Moreover, our data indicated that like class I human HDACs, Arabidopsis HDA6 was regulated by posttranslational modification. We identified for the first time that there were two specific phosphorylation sites (Ser427 and Ser429) in Arabidopsis HDA6. These two phosphorylation residues were specifically conserved in known class I HDACs, suggesting that they might be involved in the specificity for HDAC enzymatic activity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56781 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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