臺灣博碩士論文加值系統

植物組蛋白去乙醯基酶(Histone deacetylases, HDAs)參與調控植物的生長與發育。組蛋白去乙醯基酶主要作用於組蛋白上的離胺酸(Lysine)的末端，當組蛋白失去了乙醯基的修飾後，染色質濃縮，進而造成該區基因無法表現。HDA15是屬於第二群組蛋白去乙醯基酶，HDA15可與PIF3(Pytochrome interacting factor 3)交互作用並去除組蛋白H3與H4上的乙醯基修飾，進而調控葉綠素的生合成路徑。目前部分的訊息調控雖已被研究，但其酵素作用之分子機制仍不清楚。
本研究利用大腸桿菌異源系統，表達植物第二群組蛋白去乙醯基酶的去乙醯基功能區(HDA5 HD、HDA15 HD與HDA18 HD)之重組蛋白質。在蛋白質純化過程中發現HDA15 HD為四聚體且具有較高的酵素活性，經管柱液相層析實驗及生化分析，測定HDA15 HD四聚體與單體的酵素活性，同時比較HDA5與HDA18 的去乙醯基功能區，結果顯示HDA15四聚體活性最高。若將HDA15的N端123胺基酸補回HDA15 HD，其酵素活性可提高近五倍；由膠體過濾層析與高效液相層析實驗發現，HDA15 HD四聚體在加回N端後會轉變成三聚體，顯示N端具有影響酵素活性及多聚體形成的能力。若以膠體過濾層析與生化實驗分析HDA15.1與HDA15.2同形蛋白(isoforms)，結果顯示C端具有抑制酵素活性功能，並可促進HDA15多聚體的形成。HDA15 HD之結晶結構顯示為四聚體，各個單體交互作用區接近酵素活性中心，具有穩定活性中心胺基酸的能力，促使酵素催化活性上升。利用HD功能區結構比對，雖然HD核心結構相似，但HDA15 HD多聚體的形成方式多變，其影響酵素活性上升的方式有其獨特性，推測HDA15去乙醯基酶活性受其C端與其他調控因子的影響。本篇研究解析植物第一個組蛋白去乙醯基酶晶體結構，其酵素活性與多聚體形成有關，藉由此結構與功能分析，將有助於明瞭去乙醯基酶的分子調控機制。

Histone deacetylases (HDAs) involved in growth and developmental processes in plants. HDAs remove the acetyl group from Lysine residue on histone tail and cause chromatin condensation for suppressing gene expression. HDA15 is a member of class II HDA. HDA15 interacts with PIF3, a transcription factor, to repress biosynthesis of chlorophyll by reomoving acetyl modification on H3 and H4 histones. Although several study reveal HDA15 how to control physiology regulation, but its molecular mechanism is still unknown.
In this sudy, an overexpression system of Escherichia coli was used to produce recombinant protein of Histone deacetylase domain (HD domain) of all class II HDAs, including HDA5, HDA15, and HDA18. During protein purification, HDA15 HD showed much higher enzyme activity. Gel filtration chromatography revealed that HDA15 HD contained two forms of tetramer and monomer in solution, but HDA5 HD and HDA18 HD showed only monomer. In biochemical analyses, HDA15 HD tetramer showed the highest enzyme activity than those of HDA5 HD monomer, HDA15 HD monomer and HDA18 HD monomer. For restoring 123 residues of N terminus to HD of HDA15, it became to trimer and its enzyme activity was 5-fold higher than that of HDA15 HD tetramer. The results demonstated the Nt of HDA15 would affect the enzymatic activity and its oligomerization. Since two isoforms HDA15.1 and HDA15.2 with slightly difference of C-terminus existed in plants, the results from gel filtration chromatography and HPLC revealed that HDA15.1 and HDA15.2 contained no deacetylase activity and fromed aggregation. It showed HDA15 C-terminus could regulate the deacetylase activity and affect its aggregation. X-ray crystallography showed crystal structure of HDA15 HD is tetramer. The interaction interface of tetramer is nearby active site and then stabilizes the residues of active site. Finally, tetramer form could enhance its enzyme activity. By superimposition of HD structures, the overall structures of HD are similar. However, HDA15 shows its various oligomerization and regulates the enzyme activity with N-terminus and C-terminus. This is the first structure of histone deacetylase domain from plant. Its oligomerizaion showed correlation with its enzymatic activity. From structural and fuctional studies, it will provide a new insight into molecular mechanism in histone deacetylases.

中文摘要 I
Abstract III
縮寫對照表 V
圖目錄 XI
表目錄 XII
第一章 前言 1
1-1 表觀遺傳學(Epigenetics) 1
1-2 組蛋白的轉譯後修飾 1
1-3 組蛋白乙醯基轉移酶與組蛋白去乙醯基酶 2
1-3-1 阿拉伯芥中的組蛋白乙醯基轉移酶之分類與功能 2
1-3-2 阿拉伯芥中的組蛋白去乙醯基酶之分類與功能 3
1-3-3 阿拉伯芥中的Class II HDAs 4
1-3-4 HDA5(Histone deacetylase 5) 4
1-3-5 HDA15(Histone deacetylase 15) 5
1-3-6 HDA18(Histone deacetylase 18) 6
1-4 組白去乙醯基酶的蛋白質結構 6
1-5 研究方向 7
第二章 材料與方法 8
2-1 實驗材料 8
2-2 實驗方法 8
2-2-1 大腸桿菌勝任細胞製備 8
2-2-2 大腸桿菌之轉型作用 (transformation) 8
2-2-3 表現載體pET 3.1之製備與構築 8
2-2-4 阿拉伯芥Class II HDA功能區(HD)之製備與構築 9
2-2-5 重組蛋白質之大量表現 (over-expression) 與純化 10
2-2-6 SDS-聚丙烯醯胺膠體電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 11
2-2-7 高解析液相膠體過濾層析(High Performance Liquid Chromatography, HPLC) 12
2-2-8 蛋白質定量分析 12
2-2-9 HDA15 HD四聚體之蛋白質結晶 12
2-2-10 晶體繞射數據收集和處理 12
2-2-11 HDA15 HD四聚體蛋白質結構的決定與精調 13
2-2-12 去乙醯基酶之酵素動力學分析 13
2-2-13 膠體層析後之蛋白質液去乙醯基酵素活性分析 14
2-2-14 檢測植物體中HDA15之Isoform表現量多寡 14
第三章 結果 16
3-1 各種阿拉伯芥之Class II HDA重組蛋白質表現、純化、多聚體分析與去乙醯基酵素動力學檢測 16
3-1-1 HDA15 HD重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 16
3-1-2 HDA5 HD重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 16
3-1-3 HDA18 HD重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 17
3-1-4 HDA15 NtHD重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 17
3-1-5 以HPLC實驗比較阿拉伯芥之Class II HDA重組蛋白質之多聚體形式 18
3-1-6 MBP-HDA15 NtHD重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 18
3-1-7 MBP-HDA15.1-His12重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 19
3-1-8 MBP-HDA15.2-His12重組蛋白質大量表現、純化、多聚體分析與去乙醯基酵素動力學檢測 19
3-1-9 MBP-HDA15 NtHD、MBP-HDA15.1-His12與MBP-HDA15.2-His12的多聚體形式組成與活性比較 20
3-2 植物體中的HDA15 Isoform表現量之差異檢測 21
3-3 HDA15 HD四聚體蛋白質結晶結構之決定 21
3-3-1 HDA15 HD四聚體重組蛋白質結晶 21
3-3-2 X-ray繞射實驗與結構分析 21
第四章 討論 24
4-1 阿拉伯芥class II HDA之去組蛋白乙醯基酶(HD)活性比較 24
4-2 HDA15之Nt、HD與Ct功能區段之探討 25
4-3 HDA15 HD之多聚體結構與其他物種Class II HDAC結構比較 28
第五章 結論 32
參考文獻 33
圖表 38
附錄 89

陳奕睿 (2015) 阿拉伯芥第二群組蛋白去乙醯酶AtHDA5之結構及生化分析。 國立台灣大學植物科學研究所碩士論文。
Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., and Zwart, P.H. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221.
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., Faulkner, R., and Mirsky, A.E. (1964). 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.
Bottomley, M.J., Lo Surdo, P., Di Giovine, P., Cirillo, A., Scarpelli, R., Ferrigno, F., Jones, P., Neddermann, P., De Francesco, R., Steinkuhler, C., Gallinari, P., and Carfi, A. (2008). Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain. J Biol Chem 283, 26694-26704.
Boycheva, I., Vassileva, V., and Iantcheva, A. (2014). Histone acetyltransferases in plant development and plasticity. Curr Genomics 15, 28-37.
Bressi, J.C., Jennings, A.J., Skene, R., Wu, Y., Melkus, R., De Jong, R., O''Connell, S., Grimshaw, C.E., Navre, M., and Gangloff, A.R. (2010). Exploration of the HDAC2 foot pocket: Synthesis and SAR of substituted N-(2-aminophenyl)benzamides. Bioorg Med Chem Lett 20, 3142-3145.
Bunkoczi, G., and Read, R.J. (2011). Improvement of molecular-replacement models with Sculptor. Acta Crystallogr D Biol Crystallogr 67, 303-312.
Chen, W.Q., Li, D.X., Zhao, F., Xu, Z.H., and Bai, S.N. (2016). One additional histone deacetylase and 2 histone acetyltransferases are involved in cellular patterning of Arabidopsis root epidermis. Plant Signal Behav 11, e1131373.
Clapier, C.R., and Cairns, B.R. (2009). The biology of chromatin remodeling complexes. Annu Rev Biochem 78, 273-304.
Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501.
Emil Fischer. (1894). Einfluss der configuration auf die wirkung der enzyme. Eur J Inorg Chem 27, 2985-2993.
Falkenberg, K.J., and Johnstone, R.W. (2014). Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 13, 673-691.
Fina, J.P., and Casati, P. (2015). HAG3, a histone acetyltransferase, affects UV-B responses by negatively regulating the expression of DNA repair enzymes and Sunscreen Content in Arabidopsis thaliana. Plant Cell Physiol 56, 1388-1400.
Gigolashvili, T., Engqvist, M., Yatusevich, R., Muller, C., and Flugge, U.I. (2008). HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana. New Phytol 177, 627-642.
Golderer, G., and Grobner, P. (1991). ADP-ribosylation of core histones and their acetylated subspecies. Biochem J 277 ( Pt 3), 607-610.
Hollender, C., and Liu, Z. (2008). Histone deacetylase genes in Arabidopsis development. J Integr Plant Biol 50, 875-885.
Job, G., Brugger, C., Xu, T., Lowe, B.R., Pfister, Y., Qu, C., Shanker, S., Banos Sanz, J.I., Partridge, J.F., and Schalch, T. (2016). SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules. Mol Cell 62, 207-221.
Kim, B., Pithadia, A.S., and Fierke, C.A. (2015). Kinetics and thermodynamics of metal-binding to histone deacetylase 8. Protein Sci 24, 354-365.
Kornberg, R.D. (1974). Chromatin structure: a repeating unit of histones and DNA. Science 184, 868-871.
Koshland, D.E. (1958). Application of a theory of enzyme specificity to protein synthesis. Proc Natl Acad Sci U S A 44, 98-104.
Krissinel, E., and Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797.
Lauffer, B.E., Mintzer, R., Fong, R., Mukund, S., Tam, C., Zilberleyb, I., Flicke, B., Ritscher, A., Fedorowicz, G., Vallero, R., Ortwine, D.F., Gunzner, J., Modrusan, Z., Neumann, L., Koth, C.M., Lupardus, P.J., Kaminker, J.S., Heise, C.E., and Steiner, P. (2013). Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. J Biol Chem 288, 26926-26943.
Lee, J.H., Maskos, K., and Huber, R. (2009). Structural and functional studies of the yeast class II Hda1 histone deacetylase complex. J Mol Biol 391, 744-757.
Li, C., Xu, J., Li, J., Li, Q., and Yang, H. (2014). Involvement of Arabidopsis HAC family genes in pleiotropic developmental processes. Plant Signal Behav 9, e28173.
Liebich, H.M., Gesele, E., Wirth, C., Woll, J., Jobst, K., and Lakatos, A. (1993). Non-enzymatic glycation of histones. Biol Mass Spectrom 22, 121-123.
Liu, C., Li, L.C., Chen, W.Q., Chen, X., Xu, Z.H., and Bai, S.N. (2013a). HDA18 affects cell fate in Arabidopsis root epidermis via histone acetylation at four kinase genes. Plant Cell 25, 257-269.
Liu, X., Yang, S., Zhao, M., Luo, M., Yu, C.W., Chen, C.Y., Tai, R., and Wu, K. (2014). Transcriptional repression by histone deacetylases in plants. Mol Plant 7, 764-772.
Liu, X., Chen, C.Y., Wang, K.C., Luo, M., Tai, R., Yuan, L., Zhao, M., Yang, S., Tian, G., Cui, Y., Hsieh, H.L., and Wu, K. (2013b). PHYTOCHROME INTERACTING FACTOR3 associates with the histone deacetylase HDA15 in repression of chlorophyll biosynthesis and photosynthesis in etiolated Arabidopsis seedlings. Plant Cell 25, 1258-1273.
Lobera, M., Madauss, K.P., Pohlhaus, D.T., Wright, Q.G., Trocha, M., Schmidt, D.R., Baloglu, E., Trump, R.P., Head, M.S., Hofmann, G.A., Murray-Thompson, M., Schwartz, B., Chakravorty, S., Wu, Z., Mander, P.K., Kruidenier, L., Reid, R.A., Burkhart, W., Turunen, B.J., Rong, J.X., Wagner, C., Moyer, M.B., Wells, C., Hong, X., Moore, J.T., Williams, J.D., Soler, D., Ghosh, S., and Nolan, M.A. (2013). Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group. Nat Chem Biol 9, 319-325.
Luo, M., Tai, R., Yu, C.W., Yang, S., Chen, C.Y., Lin, W.D., Schmidt, W., and Wu, K. (2015). Regulation of flowering time by the histone deacetylase HDA5 in Arabidopsis. Plant J 82, 925-936.
Ma, X., Lv, S., Zhang, C., and Yang, C. (2013). Histone deacetylases and their functions in plants. Plant Cell Rep 32, 465-478.
Millard, C.J., Watson, P.J., Celardo, I., Gordiyenko, Y., Cowley, S.M., Robinson, C.V., Fairall, L., and Schwabe, J.W. (2013). Class I HDACs share a common mechanism of regulation by inositol phosphates. Mol Cell 51, 57-67.
Nathan, D., Sterner, D.E., and Berger, S.L. (2003). Histone modifications: Now summoning sumoylation. Proc Natl Acad Sci U S A 100, 13118-13120.
Nelson, C.J., Santos-Rosa, H., and Kouzarides, T. (2006). Proline isomerization of histone H3 regulates lysine methylation and gene expression. Cell 126, 905-916.
Nowak, S.J. and Corces, V.G. (2004). Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet 20, 214-220.
Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-326
Pandey, R., Muller, A., Napoli, C.A., Selinger, D.A., Pikaard, C.S., Richards, E.J., Bender, J., Mount, D.W., and Jorgensen, R.A. (2002). Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes. Nucleic Acids Res 30, 5036-5055.
Robinson, P.J., and Rhodes, D. (2006). Structure of the ''30 nm'' chromatin fibre: a key role for the linker histone. Curr Opin Struct Biol 16, 336-343.
Roth, S.Y., Denu, J.M., and Allis, C.D. (2001). Histone acetyltransferases. Annu Rev Biochem 70, 81-120.
Schuetz, A., Min, J., Allali-Hassani, A., Schapira, M., Shuen, M., Loppnau, P., Mazitschek, R., Kwiatkowski, N.P., Lewis, T.A., Maglathin, R.L., McLean, T.H., Bochkarev, A., Plotnikov, A.N., Vedadi, M., and Arrowsmith, C.H. (2008). Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity. J Biol Chem 283, 11355-11363.
Servet, C., Conde e Silva, N., and Zhou, D.X. (2010). Histone acetyltransferase AtGCN5/HAG1 is a versatile regulator of developmental and inducible gene expression in Arabidopsis. Mol Plant 3, 670-677.
Shahbazian, M.D., and Grunstein, M. (2007). Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 76, 75-100.
Stanley, J.S., Griffin, J.B., and Zempleni, J. (2001). Biotinylation of histones in human cells. Effects of cell proliferation. Eur J Biochem 268, 5424-5429.
Sterner, D.E., and Berger, S.L. (2000). Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64, 435-459.
Strahl, B.D., and Allis, C.D. (2000). The language of covalent histone modifications. Nature 403, 41-45.
Sun, Z.W., and Allis, C.D. (2002). Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418, 104-108.
Watson, P.J., Fairall, L., Santos, G.M., and Schwabe, J.W. (2012). Structure of HDAC3 bound to co-repressor and inositol tetraphosphate. Nature 481, 335-340.
West, A.C., and Johnstone, R.W. (2014). New and emerging HDAC inhibitors for cancer treatment. J Clin Invest 124, 30-39.
Wondrak, G.T., Cervantes-Laurean, D., Jacobson, E.L., and Jacobson, M.K. (2000). Histone carbonylation in vivo and in vitro. Biochem J 351 Pt 3, 769-777.
Xiao, J., Zhang, H., Xing, L., Xu, S., Liu, H., Chong, K., and Xu, Y. (2013). Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis. J Plant Physiol 170, 444-451.
Zhang, Y., and Reinberg, D. (2001). Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 15, 2343-2360.