臺灣博碩士論文加值系統

肌細胞核因子(Myocyte Nuclear Factor, MNF)是一種在老鼠體內會選擇性的表現在myogenic stem cell的轉錄因子。而肌細胞核因子所調節的基因主要在負責協調myogenic stem cell的增殖與分化，例如當肌細胞受傷時肌細胞核因子便會有大量表現的現象發生。在生物體內的肌細胞核因子可經由選擇性接合(alternative splicing)產生兩種不同的cDNA，他們各自轉譯為肌細胞核因子α及肌細胞核因子β，轉譯所得的蛋白質分別含617及414個胺基酸；他們的DNA結合區域雖是相同的，然而肌細胞核因子α及肌細胞核因子β在功能上的表現卻是完全不同的；他們在體外試驗中，對於與DNA結合的能力上有著截然不同的特徵；另外，transient-transfection assays中所表現出調控轉錄的特性也有所差異。
肌細胞核因子α、肌細胞核因子βDNA結合區域的範圍包括第289到389個殘基，且屬於winged-helix family的成員；從胺基酸的序列比對發現，肌細胞核因子之DNA結合區域與已知超過200個的winged-helix family具有35﹪~89﹪的相似性；雖然這個DNA結合區域十分具有保留性，但近來卻發現不同的winged-helix family蛋白質能以其獨特的方式辨認特異的DNA序列，繼而對各自下游的基因進行調控作用。為了釐清肌細胞核因子的DNA結合區域在結構上與功能上的關係，我們首先以E.coli系統表現出肌細胞核因子的DNA結合區域，產量大約是40~50mg/L，再利用多維異核核磁共振光譜(heteronuclear multidimensional NMR spectroscopy)推論出肌細胞核因子的DNA結合區域之三級結構；根據三級結構的分析指出，在原本應屬於第二個翅膀狀結構的區域產生了一個新的α-helix結構，這或許與winged helix family的DNA結合能力之專一性有關；另外肌細胞核因子的二級結構與間白素結合因子(ILF-DBD)很相似；綜合結構上的結果，我們比較了肌細胞核因子與ILF-DBD及其他winged-helix family的蛋白質在DNA結合能力的專一性上之不同,並且提出一些新的論點，解釋這個家族與DNA結合能力的專一性之可能原因。

Myocyte nuclear factors (MNFs) are transcription factors that are expressed selectivity in mouse myogenic stem cells. MNFs regulate the genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury. Two MNF isoforms are found, MNF-α and MNF-β each contains 617 and 414 amino acids, respectively. They are derived from a single MNF gene by alternative splicing. However, the expressions of MNF-α and MNF-β are differentially regulated. They possess distinctive functional properties with respect to DNA binding in vitro and transcriptional regulatory activity in transient—transfection assays. The DNA-binding domains of MNFs belongs to the winged helix/forkhead family since the residues from 289 to 389 of MNFs share 35﹪to 89﹪similarity with other known members of this family. Members of this family are characterized by a conserved 100-amino acid DNA-binding domain that contain threeα-helices (H1, H2, and H3), threeβ-strands (S1 and S2), and two wing-like loops (W1 and W2). Although the DNA binding domains of the winged helix family proteins are conserved, they can recognize specific DNA sequence to regulate their own downstream genes. In order to elucidate the structural and functional relationships of the DNA-binding domain of MNFs, we expressed the protein in E.coli with a yield of 40-50mg/L. We also assigned 1H, 13C and 15N resonances and deduced the secondary structure of the DNA-binding domain of MNF from multidimensional NMR spectroscopy. The secondary structures of the DNA-binding domain of MNF are similar to those of interleukin enhancer binding factor (ILF). Based on the resulting structures, their DNA-binding specificities were compared. Secondary structure analysis revealed that the wing 2 region contains an extra α-helix, which may be responsible for the DNA-binding specificity differences among members of winged helix family. This result suggests the possible structural diversity in the winged helix/forkhead proteins.

中文摘要 I
英文摘要 III
致謝 IV
目錄 V
圖目錄 VIII
表目錄 XI
縮寫檢索表 XII
儀器 XIII
第一章 緒論
1-1 肌細胞核因子( MNF )之簡介 1
1-2 肌細胞核因子DNA結合區 ( MNF-DBD ) 之功能 3
1-3 肌細胞核因子核酸結合區之結構 4
1-4 核磁共振決定蛋白質之三維結構 6
1-5 研究動機及內容簡介 8
第二章 材料與結果
2-1 MNF 的 DNA 結合蛋白之基因構築 10
2-1-1 聚合酶連鎖反應(PCR) 10
2-1-2 限制酶消化反應(digestion) 12
2-1-3 接和反應(Ligation) 13
2-1-4 形質轉換(Transformation) 13
2-2 MNF的DNA結合蛋白之表現與純化 16
2-2-1 基因的生長及誘發(Induction) 16
2-2-2 細胞萃取物之製備 18
2-2-3 以陽離子交換色層分析法之純化 18
2-2-4 逆向高效能液相層析法(Reverse-phase HPLC) 20
2-2-5 SDS-PAGE分析 21
2-2-6 Mass的測定 24
2-3 MNF-DBD蛋白三級結構之研究 25
2-3-1 利用NMR方法決定MNF-DBD的三級結構 25
2-3-2 樣品製備 26
2-3-3 Selectively 15N- labeled amino acids蛋白的製備 27
2-3-4 NMR光譜的測定 29
2-3-5 質子判定(Proton assignment) 31
2-3-6 循序判定(Sequential assignment) 31
2-3-7 分子結構的限制條件 33
2-3-8 三級結構的運算 35
第三章 實驗結果
3-1 MNF的DNA結合區域之基因構築 43
3-2 MNF的DNA結合蛋白之表現及純化 43
3-2-1 MNF的DNA結合蛋白之表現及初純化 43
3-2-2 MNF的DNA結合蛋白之再純化及SDS-PAGE分析 44
3-2-3 MNF的DNA結合蛋白之質譜分析 44
3-3 MNF-DBD之NMR圖譜的分析 45
3-3-1 蛋白質循序判定 45
3-3-2 NOE訊號的收集及整理 46
3-4 MNF-DBD二級結構的分析 47
3-4-1 Cα和Hα化學位移α 47
3-4-2 端距離NOE的訊息 47
3-4-3 MNF-DBD的二級結構 48
3-5 MNF-DBD的三級結構 50
第四章 討論
4-1 Winged-helix 家族與辨認DNA序列的研究 56
4-2 MNF-DBD、ILF-DBD、Genesis、HNF3γ的比較及分析 56
4-2-1 二級結構的比較 57
4-2-2氫鍵的分析 58
4-2-3φ角自旋自旋偶合常數的分析 58
4-2-4三級結構的比較及分析 59
4-3 Winged helix家族的分類 61
第五章 結論 63
參考文獻 65
圖 69
表 108
附錄一 118
自述 119

Boise, L. H., Petryniak, B., Mao, X., June, C. H., Wang, C., Lindstern, T., Bravo, R., Kovary, K., Leiden, J. M., and Thompson, C. B. (1993). The NFAT-1 DNA binding complex in activated T cells contains Fra-1 and JunB, Mol. Cell. Biol., 13: 1911.
Clark, K. L., Halay, E. D., Lai, E., and Burley, S. K. (1993). Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5, Nature, 364: 412.
Daniel, J. G., Quan, Y., Rhonda, B. D., and R. Sanders, W. (1997). Persistent expression of MNF identifies myogenic stem cells in postnatal muscles, Developmental Biology, 188: 280.
Daniel, J. G., Annette, M., Joel, E., Yuhe, Z., Peng Y., Rhonda, B. D., and R. Sanders W. (1999). Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF, Biochem. J., 345: 335.
Eckhard K., and Walter, K. (1996). Five years on the wings of fork head, Mechanism of Development, 57: 3.
Gajiwala, K. S., and Burley, S. K. (2000). Winged helix proteins, Curr. Opin. Struct. Biol., 10: 110.
Gajiwala, K. S., Chen, H., Comille, F., Roques, B. P., Reith, W., Mach, B., and Burley, S. K. (2000). Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding, Nature, 403: 916.
Garry DJ, Meeson A, Elterman J, Zhao Y, Yang P, Bassel-Duby R,Williams RS., Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF. (2000) Proc Natl Acad Sci U S A, 10 5416
Hong, W., Lawrence, P. M., and Barbara, J. G. (2002). Inhibitory module of Ets-1 allosterically regulates DNA binding through a dipole-facilitated phosphate contact, J. Biol. Chem., 277: 2225.
Hromas, R., Ye, H., Spinella, M., Dmitrovsky, E., Xu, D., and Costa, R. H. (1999). Genesis, a winged helix transcriptional repressor, has embryonic expression limited to the neural crest, and stimulates proliferation in vitro in a neural development model, Cell Tissue Res., 297: 371.
Ian, M., Changwen, J., and Xiubei, L. (1999). Structural changes in the region directly adjacent to the DNA-binding helix highlight a possible mechanism to explain the observed changes in the sequence-specific binding of winged helix proteins, J. Mol. Biol., 289: 683.
Jin, C., and Liao, X. (1999). Backbone dynamics of a winged helix protein and its DNA complex at different temperatures: changes of internal motions in genesis upon binding to DNA, J. Mol. Biol., 292: 641.
Jin, C., Marsden, I., Chen, X., and Liao, X. (1999). Dynamic DNA contacts observed in the NMR structure of winged helix protein-DNA complex, J. Mol. Biol., 289: 683.
Jin, C., Marsden, I., Chen, X., and Liao, X. (1998). Sequence specific collective motions in a winged helix DNA binding domain detected by 15N relaxation NMR, Biochem, 37: 6179.
John, C., Wayne, J. F., Arthur G. P., and Nicholas J. S. (1996). Protein NMR Spectroscopy.
Joon, Y. L., Zhuqing, Q. P., Baohong, C., Shigemi, K., Ron, J., James, C., Arvydas, U., Charley, G., Paul, R., Anton, W., and Johnny, H. (2000). Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing, J. Cell Biol., 150: 1085.
Li, C., Lai, C. F., Sigman, D. S. and Gaynor, R. B. (1991). Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat, Proc. Natl. Acad. Sci. USA, 88: 7739.
Marsden, I., Jin, C., and Liao, X. (1998). Structural changes in the region directly adjacent to the DNA-binding helix highlight a possible mechanism to explain the observed changes in the sequence-specific binding of winged helix proteins, J. Mol. Biol., 278: 293.
Marsden, I., Chen, Y., Jin, C., and Liao, X. (1997). Evidence that the DNA binding specificity of winged helix proteins is mediated by a structural change in the amino acid sequence adjacent to the principal DNA binding helix, Biochem, 36: 13248.
Nirula, A., Moore, D. J., and Gaynor, R. B. (1997). Constitutive binding of the transcription factor interleukin-2 (IL-2) enhancer binding factor th the IL-2 promoter, J. Biol. Chem., 272: 7736.
Pei-Phen L., Yen-Chin C., Chin L., Yu-Huei H., Shu-Wan C., Shu-Huei C., Wen-Yih J., and Woei-Jer C., (2002) Solution Structure of the DNA-Binding Domain of Interleukin Enhancer Binding Factor (FOXK1), the Forkhead Transcription Factor with an Extra C-Terminal Helix, Proteins.
Quan, Y., Yanfeng, K., Beverly, R., Daniel, J. G., Rhonda, B. D., and R. Sanders, W. (2000). The winged-helix/forkhead protein myocyte nuclear factor β (MNF-β) forms a co-repressor complex with mammalian Sin3B, J. Mol. Biol., 127: 5213.
Quan, Y., Rhonda, B. D., and R. Sanders, W. (1997). Transient expressiom of a winged-helix protein, MNFβ, during myogenesis, Mol. Cell. Biol., 17: 5236.
Siekecitz, M., Josephs, S. F., Dukovich, M., Peffer, N., Wong-Staal, F., and Greene, W. C. (1987). Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I, Science, 238: 1575.
Wanyun, S., Mark, R., and Xiubei, L. (2002). Structure comparison of two conserved HNF-3/fkh proteins HFH-1 and genesis indicates the existence of folding difference in their complexes with a DNA binding sequence, Biochem., 41: 3286.
Weigel, D., and Jackle, H. (1990). The fork head domain: a novel DNA binding motif of eukaryotic transcription factors, Cell, 63: 455.
Wuthrich, K. (1986). NMR of Proteins and Nucleic Acids.
陳金榜，(1994)，蛋白質在水溶液中之結構-多維異核核磁共振之應用， 科儀新知，第十五卷，第六期，40-46。
黃忠智及余靖，(1994)，利用核磁共振光譜決定台灣眼鏡蛇蛇毒蛋白分子的水溶液中三度空間結構，科儀新知，第十五卷，第六期，21-39。
謝玉惠，(1999)，間白素結合因子(ILF)核酸結合區的功能及其結構之研究，國立成功大學生物化學研究所碩士論文。
劉沛棻，(2001)，利用核磁共振光譜決定間白素結合因子(ILF)核酸結合區的水溶液中三度空間結構，國立成功大學生物化學研究所碩士論文。