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標題: | 阿拉伯芥 HDA15 組蛋白去乙醯基酶功能區結構及其受磷酸化調控機制之研究 Crystal Structure of Arabidopsis HDA15 Histone Deacetylase Domain and Its Enzymatic Activity Regulated by Phosphorylation |
作者: | Heng-Chen Hung 洪珩宸 |
指導教授: | 鄭貽生 |
關鍵字: | 去乙醯基?, deacetylase, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 阿拉伯芥組蛋白去乙醯基酶15 (Histone deacetylase 15, HDA15)是屬於第二群組蛋白去乙醯基酶,主要表現在細胞核中,有部分研究已了解HDA15酵素活性及其多聚體形式,也發現到HDA15第448號與第452號的絲胺酸(Serine, S)被磷酸化後,會影響去乙醯基能力,但並未深入探討HDA15活性變化與分子機制,本篇將HDA15 HD (Histone deacetylase domain)以及ZFHD (Zinc finger domain and histone deacetylase domain)構築在表現載體內並大量表現於大腸桿菌中。
實驗結果以解析度2.36 Å蛋白質晶體得到了HDA15 HD四聚體結構,分析出了HD四聚體之每個單體是由12個α- helix與9個β- sheets所組成的,而且每個HD單體中都含有一個鋅離子並確定了鋅離子之位置所在;同時也得到了HDA15 HD四聚體與TSA (Trichostatin A)抑制劑複合體結構,當TSA進入了活性口袋會與鋅離子形成離子鍵結,使TSA穩定停留在活性口袋中,讓乙醯基無法進入而抑制了去乙醯基酶的活性。另外,原本HDA15 HD會以高活性的四聚體和極低活性的單體兩種形式呈現,如果把N端的鋅手指功能區加回序列中成為ZFHD,就會變成一個單獨二聚體形式,而且活性會比HD四聚體還要高大約10倍,可以知道鋅手指功能區能有效地促進受質進入活性口袋中,此外也可能對HDA15 ZFHD穩定形成二聚體結構具有一定的影響力。接著利用模擬磷酸化的方式,將HDA15 HD與ZFHD的Ser448和Ser452突變成天冬胺酸(Aspartate, D),表現結果都只會以單體形式呈現,而被磷酸化的去乙醯基酶活性都會降低到跟原來HDA15 HD單體差不多的狀態;相反的,若將Ser448和Ser452突變成丙胺酸(Alanine, A),不但形式變回四聚體,酵素活性也恢復到原本的狀態,證明了HDA15的酵素活性會受到Ser448和Ser452位點磷酸化調控,並藉由多聚體以及單體兩種形式機制進行活性的轉換。最後,本篇以更好的解析度修正了HDA15 HD四聚體結構,也更進一步研究酵素活性高低及穩定性與磷酸化調控、形式變化有關,期望本研究能有助於更了解HDA15的分子機制。 Histone deacetylase 15 (HDA15) of Arabidopsis thaliana is classified to class II in RPD3/HDA1-like superfamily. The phosphorylated residues of HDA15 were identified at Ser448 and Ser452, and the phosphorylation cause to reduce the activity of deacetylase. However, the phosphorylated enzymatic analysis and the molecular mechanism of HDA15 remain unknown. Here, the recombinant proteins of HDA15 histone deacetylase domain (HD) and zinc finger domain histone deacetylase domain (ZFHD) were overexpressed in E.coli. In this study, the crystal resolution of HDA15 HD tetramer which is better than before is resolved at 2.36 Å. Each HD monomer is composed of 12 α-helices, 9 β-sheets and a zinc ion. The zinc ion coordinates the side chains of Asp313, His315 and Asp404 by forming salt briage in the active pocket. Additionally, the co-crystal structure of HDA15 HD tetramer with Trichostatin A (TSA) complex is resolved. Owing to the fact that serrated terminus of TSA in the active site could stably form an ionic bond with the zinc ion, the acetyl group couldn’t enter the cavity and inhibit the activity of histone deacetylase. Gel filtration chromatography reveals that HDA15 HD contains two forms, higher activity tetramer and lower activity monomer, in solution. For restoring 86 to 115 residues zinc finger domain of N-terminus to HD, it becomes to ZFHD dimer alone and its enzymatic activity is ten-fold higher than that of HD tetramer. The results demonstate that the zinc finger domain of HDA15 can strongly promote the substrates to enter the cavity and affects to stably form dimer. Furthermore, phosphomimetic mutation of Ser448 or Ser452 to Asp are purified to reveal only monomer in solution, and phosphorylated deacetylase activity is as low as activity of wild type HD monomer. In contrast, mutation of Ser448 or Ser452 to Ala could form tetramer and restore its enzymatic activity. It shows that the enzymatic activity of HDA15 could be regulated by phosphorylation of Ser448 and Ser452 with the exchange of monomer and tetramer. From the crystal structure and the enzymatic studies, it will provide a new insight on molecular mechanism in histone deacetylases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59591 |
DOI: | 10.6342/NTU201700719 |
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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