阿拉伯芥第二群組蛋白去乙醯基酶AtHDA5之結構及生化分析

Structural and Biochemical Assays of ClassII AtHDA5

陳奕睿(Yi-Jui Chen)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02020

摘要

組蛋白去乙醯基酶(Histone Deacetylases, HDACs)主要作用為去除細胞組蛋白乙醯基，改變染色質與DNA纏繞的鬆緊程度，進而調控基因的表現。去乙醯基後之組蛋白，電性由負電回復帶正電，使組蛋白與DNA纏繞變得較緊密，造成基因不利於表現，屬節制基因表現的角色。
阿拉伯芥組蛋白去乙醯基酶(AtHDA5)屬酵母菌RPD3家族第二群，皆具有一高度保守的組蛋白去乙醯基酶區域，AtHDA5序列的N端第10到第26個胺基酸為預測之核定位訊號(Nuclear localization signal)；C端區域則為低度保守區域。因植物組蛋白去乙醯基酶在蛋白質分子層次研究不多，其相關機制及結構仍有許多值得探究的課題。
本研究針對重組蛋白質AtHDA5，連接MBP (Maltose binding protein) 標誌分子進行蛋白質表現與純化；膠體過濾層析法與動態光散射(Dynamic light scattering)結果顯示，AtHDA5在水溶液中，具有可溶多聚體(polysome)與單體(monomer)二種不同形式；酵素活性分析，利用Boc-Lys(Ac)-AMC 作為受質，確認多聚體與單體皆具有酵素活性功能；為探究AtHDA5是否具有磷酸化位點，分別以去除N端26個胺基酸(ND26)、雙突變S7/8A及單突變S7A，其酵素活性分別下降26%到33%；模擬磷酸化位點S7/8D，可回復15%，顯示磷酸化位點對AtHDA5的HD domain具調控能力；去除C端(DCT, 385-661 a.a.)序列，去乙醯酶活性亦同時消失。次細胞定位(subcellular localization)實驗顯示，WT(含NLS)與ND26(不含NLS)的蛋白質，都僅位於細胞質中。本研究另以電子顯微鏡負染技術，初步重建MBP-AtHDA5的低解析度(~18Å)單體結構；蛋白質結構模擬，以Human HDAC4 (pdbid: 2VQO)模板，建立AtHDA5之去乙醯基酶區域之模擬結構，同時模擬受質Boc-Lys(Ac)-AMC於HD domain之結構。期望藉由對AtHDA5的分子結構與功能分析，提供未來在植物組蛋白去乙醯基酶的進一步分子機制研究。

關鍵字

阿拉伯芥 ； 組蛋白去乙醯基酶 ； 電子顯微鏡影像重建

並列摘要

Histone Deacetylases (HDACs) play a role to remove acetyl group on the histone in the cell and alter interaction between histone and DNA in chromatin to condense for regulating the gene expression, which is a kind of suppressors to repressthe transcription of genes.
Arabidopsis thaliana histone deacetylase 5 (AtHDA5), a homolog of yeast RPD3 class II, contains the histone deacetylase (HD) conserved domain. The N terminal of AtHDA5 from 10th -26th was predicted as nuclear localization signal (NLS) and the C terminal showed highly variable region. Since the plant histone deacetylases remained undiscovered in molecular levels, it worths to study the molecular function and protein structures.
In this study, we aimed to construct the recombinant AtHDA5 with a strategy from MBP (Maltose binding protein) fusion tag for protein expression and purification. Interestingly, AtHDA5 showed two different sizes of polysome and monomer form after the analyses of size-exclusion chromatography and dynamic light scattering. To confirm the enzymatic function, Boc-Lys(Ac)-AMC substrate was used to check the activity of polysome and monomer form. To identify the phosphorylation sites, the mutants of AtHDA5 ND26, AtHDA5 S7/8A and AtHDA5 S7A were prepared and showed the enzymatic activity decreased with 26%-33% compared to wildtype. The mutant with mimic phosphorylation S7/8D could rescue the 15% activity from S7/8A. The results showed that phosphorylation in AtHDA5 would regulate the enzyme activity. Truncated C-terminal (residues 385-661) caused the loss of enzyme activity of histone deacetylase. Subcelluar localization confirmed AtHDA5 located at cytoplasm.
Transmission electron microscopy was used to reconstruct a low resolution of 3D model of AtHDA5. The preliminary structure of MBP-AtHDA5 shows a monomeric form (~18 Å). A homology modeling of AtHDA5 was generated with the template of human HDAC4 (pdbid: 2VQO). Furthermore, the AtHDA5 model was used to reveal the substrate Boc-Lys(Ac)-AMC in the binding pocket of AtHDA5. The structural and functional analyses would provide the insight into the molecular mechanisms for regulating gene expression of plants.

並列關鍵字

Histone Deacetylases ； HDACs ； HDA ； AtHDA5 ； EMAN2 ； 3D reconstruction ； pMALH12-c5V ； TEV

參考文獻

Alinsug, M.V., Yu, C.W., and Wu, K. (2009). Phylogenetic analysis, subcellular localization, and expression patterns of RPD3/HDA1 family histone deacetylases in plants. BMC Plant Biol 9: 37.

Alinsug, M.V., Chen, F.F., Luo, M., Tai, R., Jiang, L., and Wu, K. (2012). Subcellular localization of class II HDAs in Arabidopsis thaliana: nucleocytoplasmic shuttling of HDA15 is driven by light. PLoS One 7: e30846.

Allfrey, V.G., Littau, V.C., and Mirsky, A.E. (1964a). Methods for the Purification of Thymus Nuclei and Their Application to Studies of Nuclear Protein Synthesis. J Cell Biol 21: 213-231.

Allfrey, V.G., Faulkner, R., and Mirsky, A.E. (1964b). Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis. Proc Natl Acad Sci U S A 51: 786-794.

Arabidopsis Genome, I. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796-815.