阿拉伯芥組蛋白去乙醯基酶HDA15多聚體與酵素活性之關鍵胺基酸研究

Study on the key residues of oligomerization and enzyme activity of Arabidopsis Histone Deacetylase 15

劉庭君(Ting-Chun Liu)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/生命科學系/碩士(2019年)

https://doi.org/10.6342/NTU201903581

摘要

阿拉伯芥組蛋白去乙醯基酶15 (Histone deacetylase 15, HDA15)為RDP3/HDAC1-like家族中的第二群組蛋白去乙醯基酶，HDA15去乙醯基功能區 (histone deacetylase domain, HD)之四聚體結構已由本實驗室解析完成，研究指出去乙醯基功能區的HDA15重組蛋白 (HD)以四聚體及單元體形式存在，而具有鋅手指區 (zinc finger domain)的去乙醯基功能區的HDA15重組蛋白 (ZFHD)會以二聚體形式存在，ZFHD二聚體酵素活性(kcat)較HD四聚體形式高40倍；另外HDA15之Ser448及Ser452為磷酸化位點，在in vitro或in vivo之實驗中，HDA15被磷酸化後，會改以單元體形式存在並造成酵素活性消失；由於HDA15磷酸化造成多聚體與活性改變的關連性尚未被探討，因此本研究以點突變分析位於二聚體及四聚體介面之胺基酸，結合酵素動力學及蛋白質結構分析，探討HDA15多聚體之形成與酵素活性的關連性。本研究針對三區進行點突變，1) loop1中央之單點突變E163A、E165A僅影響HD四聚體的形成而不影響ZFHD二聚體構型，同樣於loop1中央之單點突變Q169A皆不影響HD及ZFHD之多聚體形成；2)位於β2之Y228與Y230雙突變造成ZFHD以單元體形式存在而無法形成二聚體；3)單點突變近α2之loop1上E173A及α2上的R177A，兩者皆位在二聚體介面及活性位點，同時造成ZFHD無法形成二聚體以及活性下降。分析S448及S452磷酸化位點、去乙醯基酶活性區與二聚體在結構上的氫鍵網絡，可以發現磷酸化位點與去乙醯基酶活性區之胺基酸，以直接或間接方式連結至loop1或β2，本研究綜合胺基酸位點突變數據及氫鍵網絡分析證明HDA15之酵素活性與多聚體之形成可受到磷酸化調節之能力。

關鍵字

阿拉伯芥 ； 組蛋白去乙醯基酶 ； HDA15 ； 多聚體形成

並列摘要

Arabidopsis histone deacetylase 15 (HDA15) belongs to the class II of histone deacetylase in the RDP3/HDAC1-like family. The tetrameric structure of HDA15 HD has been resolved by our colleagues. Previous study revealed that different oligomerization states of histone deacetylase domain (HD) and zinc finger with histone deacetylase domain (ZFHD) could be observed. HDA15 HD contains tetramer and monomer while ZFHD is dimer. The enzyme activity (kcat) of ZFHD dimer is higher 40-foldthan that of HD tetramer. Moreover, two phosphorylation sites S448 and S452 with phosphomimetic would form monomer and result in loss of HDAC activity in vitro and in vivo. It is unknown about how to regulate oligomerization and enzyme activity of HDA15 by phosphorylation. Therefore, the study used site-directed mutagenesis of the key residues at the dimer and tetramer interface to explore the relationship between oligomerization and enzyme activity of HDA15. Three regions for point mutation are difined: 1) the single mutants E163A and E165A at loop1 would disrupt the formation of HD tetramer, but didn’t affect the dimer of ZFHD. The single mutant Q169A at loop 1 didn’t show any alterations. 2) The double mutant of Y228A/Y230A at β2 caused ZFHD from dimer to monomer. 3) The single mutants E173A at loop1 adjacent α2 and R177A at α2 located between the dimer interface and the active site would simultaneously disrupt oligomerization and enzyme activity. Analysis of phosphorylation sites (S448 and S452), active site, and dimer interface revealed that the loop1 and β2 are connected by several hydrogen bonds directly and indirectly. Therefore, the study provided the evidences to show how the enzyme activity and oligomerization of HDA15 could be regulated by phosphorylation via the hydrogen bond network.

並列關鍵字

Arabidopsis ； Histone Deacetylase ； HDA15 ； oligomerization

參考文獻

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