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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Tzu-Ying Chuang | en |
dc.contributor.author | 莊子瑩 | zh_TW |
dc.date.accessioned | 2021-06-17T02:18:54Z | - |
dc.date.available | 2017-09-12 | |
dc.date.copyright | 2017-09-12 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-21 | |
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J Cell Mol Med 7, 113-126 (2003). 9 Pegg, A. E. Mammalian polyamine metabolism and function. IUBMB Life 61, 880-894, doi:10.1002/iub.230 (2009). 10 Wallace, H. M. & Fraser, A. V. Inhibitors of polyamine metabolism: review article. Amino Acids 26, 353-365, doi:10.1007/s00726-004-0092-6 (2004). 11 Nowotarski, S. L., Woster, P. M. & Casero, R. A., Jr. Polyamines and cancer: implications for chemotherapy and chemoprevention. Expert Rev Mol Med 15, e3, doi:10.1017/erm.2013.3 (2013). 12 Kusano, T., Berberich, T., Tateda, C. & Takahashi, Y. Polyamines: essential factors for growth and survival. Planta 228, 367-381, doi:10.1007/s00425-008-0772-7 (2008). 13 (ed.), K. I. Regulatory Nascent Polypeptides. doi: 10.1007/978-4-431-55052-5_12 (2014). 14 Casero, R. A., Jr. & Marton, L. J. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov 6, 373-390, doi:10.1038/nrd2243 (2007). 15 Almrud, J. J. et al. Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding. J Mol Biol 295, 7-16, doi:10.1006/jmbi.1999.3331 (2000). 16 Pegg, A. E. Regulation of ornithine decarboxylase. J Biol Chem 281, 14529-14532, doi:10.1074/jbc.R500031200 (2006). 17 Jackson, L. K., Brooks, H. B., Osterman, A. L., Goldsmith, E. J. & Phillips, M. A. Altering the reaction specificity of eukaryotic ornithine decarboxylase. Biochemistry 39, 11247-11257 (2000). 18 Shantz, L. M. & Pegg, A. E. Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway. Int J Biochem Cell Biol 31, 107-122 (1999). 19 Erales, J. & Coffino, P. Ubiquitin-independent proteasomal degradation. Biochim Biophys Acta 1843, 216-221, doi:10.1016/j.bbamcr.2013.05.008 (2014). 20 Jariel-Encontre, I., Bossis, G. & Piechaczyk, M. Ubiquitin-independent degradation of proteins by the proteasome. Biochim Biophys Acta 1786, 153-177, doi:10.1016/j.bbcan.2008.05.004 (2008). 21 Rajput, B., Murphy, T. D. & Pruitt, K. D. RefSeq curation and annotation of antizyme and antizyme inhibitor genes in vertebrates. Nucleic Acids Res 43, 7270-7279, doi:10.1093/nar/gkv713 (2015). 22 Samal, K. et al. AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport. Int J Cancer 133, 1323-1333, doi:10.1002/ijc.28139 (2013). 23 Coffino, P. Antizyme, a mediator of ubiquitin-independent proteasomal degradation. Biochimie 83, 319-323 (2001). 24 Heller, J. S., Fong, W. F. & Canellakis, E. S. Induction of a protein inhibitor to ornithine decarboxylase by the end products of its reaction. Proc Natl Acad Sci U S A 73, 1858-1862 (1976). 25 Olsen, R. R. & Zetter, B. R. Evidence of a role for antizyme and antizyme inhibitor as regulators of human cancer. Mol Cancer Res 9, 1285-1293, doi:10.1158/1541-7786.MCR-11-0178 (2011). 26 Newman, R. M. et al. Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression. J Biol Chem 279, 41504-41511, doi:10.1074/jbc.M407349200 (2004). 27 Lim, S. K. & Gopalan, G. Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A. Oncogene 26, 6593-6603, doi:10.1038/sj.onc.1210482 (2007). 28 Gruendler, C., Lin, Y., Farley, J. & Wang, T. Proteasomal degradation of Smad1 induced by bone morphogenetic proteins. J Biol Chem 276, 46533-46543, doi:10.1074/jbc.M105500200 (2001). 29 Kasbek, C., Yang, C. H. & Fisk, H. A. Antizyme restrains centrosome amplification by regulating the accumulation of Mps1 at centrosomes. Mol Biol Cell 21, 3878-3889, doi:10.1091/mbc.E10-04-0281 (2010). 30 Tajima, A. et al. Polyamine regulating protein antizyme binds to ATP citrate lyase to accelerate acetyl-CoA production in cancer cells. Biochem Biophys Res Commun 471, 646-651, doi:10.1016/j.bbrc.2016.02.084 (2016). 31 Hoshino, K. et al. Polyamine transport by mammalian cells and mitochondria: role of antizyme and glycosaminoglycans. J Biol Chem 280, 42801-42808, doi:10.1074/jbc.M505445200 (2005). 32 Hoffman, D. W., Carroll, D., Martinez, N. & Hackert, M. L. Solution structure of a conserved domain of antizyme: a protein regulator of polyamines. Biochemistry 44, 11777-11785, doi:10.1021/bi051081k (2005). 33 Kurian, L., Palanimurugan, R., Godderz, D. & Dohmen, R. J. Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature 477, 490-494, doi:10.1038/nature10393 (2011). 34 Matsufuji, S. et al. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80, 51-60 (1995). 35 Rom, E. & Kahana, C. Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. Proc Natl Acad Sci U S A 91, 3959-3963 (1994). 36 Baranov, P. V. et al. RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression. Nucleic Acids Res 29, 264-267 (2001). 37 Atkins, J. F., Loughran, G., Bhatt, P. R., Firth, A. E. & Baranov, P. V. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use. Nucleic Acids Res 44, 7007-7078, doi:10.1093/nar/gkw530 (2016). 38 Wu, H. Y. et al. Structural basis of antizyme-mediated regulation of polyamine homeostasis. Proc Natl Acad Sci U S A 112, 11229-11234, doi:10.1073/pnas.1508187112 (2015). 39 Hayashi, S. & Murakami, Y. Rapid and regulated degradation of ornithine decarboxylase. Biochem J 306 ( Pt 1), 1-10 (1995). 40 Zhang, M., Pickart, C. M. & Coffino, P. Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate. EMBO J 22, 1488-1496, doi:10.1093/emboj/cdg158 (2003). 41 Fujita, K., Murakami, Y. & Hayashi, S. A macromolecular inhibitor of the antizyme to ornithine decarboxylase. Biochem J 204, 647-652 (1982). 42 Murakami, Y., Ichiba, T., Matsufuji, S. & Hayashi, S. Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase. J Biol Chem 271, 3340-3342 (1996). 43 Albeck, S. et al. Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function. Protein Sci 17, 793-802, doi:10.1110/ps.073427208 (2008). 44 Bercovich, Z. & Kahana, C. Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is inhibited by antizyme. J Biol Chem 279, 54097-54102, doi:10.1074/jbc.M410234200 (2004). 45 Qiu, S., Liu, J. & Xing, F. Antizyme inhibitor 1: a potential carcinogenic molecule. Cancer Sci 108, 163-169, doi:10.1111/cas.13122 (2017). 46 Kim, S. W. et al. Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism. J Cell Sci 119, 2583-2591, doi:10.1242/jcs.02966 (2006). 47 Mangold, U., Hayakawa, H., Coughlin, M., Munger, K. & Zetter, B. R. Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification. Oncogene 27, 604-613, doi:10.1038/sj.onc.1210685 (2008). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68367 | - |
dc.description.abstract | 多胺 (polyamine) 是一類帶有多價正電性的小分子脂肪族化合物,廣泛地存在於自然界中,無論原核及真核細胞中都能觀察到多胺的存在。多胺的多價正電性使其能夠與一些帶負電分子,例如DNA、RNA和磷脂等,或是與蛋白質表面帶有負電性的區域產生可逆的靜電交互作用 (electrostatic interaction) ,進而能參與在生物體中許多重要的生理功能調控,例如:細胞生長、增殖及調節細胞分化等。多胺廣泛地影響了細胞的生長、生存與凋亡,許多研究都證實了多胺濃度異常與正常細胞癌化具有密切相關性,也因此,多胺於細胞中的濃度必須受到嚴密的調控,保持在一定範圍內的恆定,才能使細胞維持正常的生理功能。
在多胺的生合成路徑中,鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 是催化多胺合成的第一步驟,也是整個生合成反應中的速率決定步驟,其在調控多胺於胞內的濃度上,扮演一個相當重要的角色。ODC是第一個被發現不需要經泛素化 (ubiquitination) 就能被26S 蛋白酶體 (proteasome) 降解的蛋白,此降解機制被認為是調控ODC蛋白表現量最主要的方式。在此降解機制中,需要一特別調控蛋白的參與,名為抗酶 (antizyme, Az),Az的單體能夠透過競爭的方式與ODC單體形成異質二聚體 (heterodimer),抑制ODC活性,並進一步使其被26S 蛋白酶體所降解;而抗酶抑制因子 (antizyme inhibitor, AzIN) 則是另一個參與在此調控機制中的重要調控蛋白,其與Az具有截然不同的功能,AzIN會與ODC競爭和Az的結合,抑制Az的作用,藉由Az與AzIN相互拮抗,能使整個調控機制的運作更加精準。 過去我們實驗室已解出 Az-ODC的複合體結構,使我們對於Az調控ODC的機制有一定程度的了解,然而受限於先前所發表的Az-AzIN複合體結構解析度不足的緣故 (~5.8 Å) ,我們對於Az及AzIN之間的交互作用細節依舊不甚了解。本研究的目標即在於得到品質更好的晶體、解析出Az-AzIN複合體更精確的結構,以了解二者交互作用細節,就策略而言本研究企圖以跨物種間蛋白結構與功能具高度保留性的概念來突破晶體解析度不佳的困境。 我們實驗室過去的研究發現,人類AzIN (hAzIN) 與Az進行交互作用的界面胺基酸與小鼠AzIN (mAzIN) 的胺基酸有高度保留性及相似性,因此我們期待透過得到小鼠AzIN與人類Az (hAz) 所形成的晶體,藉此改善解析度不佳的問題,了解 Az與AzIN之間的交互作用細節及可能的作用機制。 在本研究中,我們建立了表現及純化mAzIN-hAz95-228蛋白複合體的系統,利用鎳離子親和性管柱、陰離子交換樹脂及膠體過濾層析法得到高濃度及高純度的mAzIN-hAz95-228蛋白複合體。目前我們已找到幾個能夠成功使mAzIN-hAz95-228蛋白複合體形成晶體的養晶條件,然而卻還無法得到高解析度的X-ray繞射圖譜,在未來我們將嘗試改善這些晶體的繞射情形,利用不同的蛋白晶體冷凍保護方法,也持續篩選新的養晶條件。 | zh_TW |
dc.description.abstract | Polyamines are small multivalent organic polycations ubiquitously present in eukaryotic cells. These compounds play multiple regulatory roles in mammalian physiology owing to their polycationic characteristics. For example, polyamines can bind to proteins and nucleic acids via electrostatic interactions and modulate their structure and functions, in turn affecting many cellular processes, including cell growth and differentiation. It has been shown that intracellular polyamine homeostasis is important for cells to maintain normal physiological functions. Aberrant accumulation of polyamine is associated with cell transformation and tumorigenesis. Therefore, intracellular polyamine level must be under tight control.
Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the rate-limiting step in the polyamine biosynthesis pathway. The activity and stability of this homodimeric protein is controlled by the regulatory protein antizyme (Az). Abundance of full-length Az is increased in response to high intracellular polyamine level through the polyamine-induced translational +1 frameshifting during the translation of Az mRNA. Az can disrupt the formation of active ODC homodimers by forming a tight 1:1 binary complex with ODC monomer, which not only inhibits catalytic activity of ODC, but triggers ODC degradation via the 26S proteasome in an ubiquitin-independent manner. Furthermore, Az can also inhibit polyamine uptake into cells, which reduce the intracellular polyamine level. Therefore, Az is regarded as a negative regulator of cellular polyamines. Antizyme inhibitor (AzIN) is another major regulatory protein involved in maintaining polyamine homeostasis. In contrast to Az, AzIN functions as a positive regulator of polyamine levels. AzIN competes with ODC for binding to Az. The formation of Az-AzIN heterodimer leads to the release of ODC and effectively replenishes ODC activity. Thus, both AZ and AzIN are important parts of the auto-regulatory circuits designed to maintain optimal levels of polyamine within a cell. To understand the structural details regarding the formation of Az-AzIN complex, our laboratory has determined the crystal structure of a truncated Az95-228 in complex with ODC and obtained a lower resolution 5.8 Å crystal structure of Az110-228-AzIN. The major objective of this work is to obtain a crystal structure of Az-AzIN at higher resolution and elucidate the interactions between Az and AzIN in atomic detail. To achieve this goal, we tested whether human Az (hAz) can complex with mouse AzIN (mAzIN), and whether the resultant complex can be crystallized for structural characterization. According to the previous studies of our laboratory, we have found that the interface residues of human and mouse AzIN are highly conserved. We have successfully built a protein expression and purification system that can be used to produce large amount of highly purified hAZ-mAzIN complex. Specifically, purification was conducted using immobilized metal affinity, ion exchange and gel filtration chromatography. Moreover, we have successfully identified several conditions by which hAz95-228-mAzIN can be crystallized. However, these crystals diffract only to low resolution at the moment. We will examine whether the diffraction quality of hAz95-228-mAzIN crystals can be further improved. Also, we will continue to search for new crystallization conditions in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:18:54Z (GMT). No. of bitstreams: 1 ntu-106-R04442010-1.pdf: 3432351 bytes, checksum: 6877c9b4c7cf725f385b6eb6ad6003ba (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 謝誌 I
摘要 II Abstract IV 縮寫表 VII 目錄 VIII 圖目錄 X 表目錄 XI 一、前言 1 1.1. 多胺 (polyamine) 1 1.1.1. 多胺之生理功能及重要性 1 1.1.2. 多胺之生合成路徑 3 1.1.3. 多胺之分解代謝路徑 3 1.1.4. 胞內多胺含量之調控 4 1.2. 鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 5 1.2.1. 鳥胺酸脫羧酶之結構 5 1.2.2. 鳥胺酸脫羧酶之生理功能與催化機制 5 1.2.3. 鳥胺酸脫羧酶之調控 6 1.3. 抗酶 (antizyme, Az) 7 1.3.1. 抗酶之生理功能與重要性 7 1.3.2. 抗酶之構形 8 1.3.3. 抗酶之調控 8 1.2.4. 抗酶與鳥胺酸脫羧酶之交互作用及降解機制 9 1.4. 抗酶抑制因子 (antizyme inhibitor, AzIN) 10 1.4.1. 抗酶抑制因子之結構 10 1.4.2. 抗酶抑制因子之生理功能與重要性 10 1.4.3. 抗酶抑制因子與抗酶之交互作用 11 1.5. 研究目的 12 二、材料與方法 13 2.1. 蛋白質表現系統 13 2.1.1. 表現質體建構 13 2.1.2. 表現蛋白菌株 13 2.1.3. 製備勝任細胞 (competent cell) 14 2.1.4. 轉型作用 (transformation) 14 2.1.5. hAz-mAzIN蛋白複合體之共表達 14 2.2. 蛋白質純化 16 2.2.1. 破菌與蛋白萃取 16 2.2.2. 液相層析 (liquid chromatography) 17 2.3. 蛋白質濃縮及定量 20 2.3.1. 膠體電泳分析 (gel electrophoresis) 20 2.3.2. mAzIN-hAz95-228複合體濃縮 21 2.3.2. 定量mAzIN-hAz95-228複合體 22 2.4. 蛋白質晶體培養 22 2.4.1. 蛋白結晶之晶體培養方法 22 2.4.2. 蛋白結晶之條件初步篩選 23 2.4.3. 蛋白結晶之條件微調 24 2.6. 蛋白質晶體之X-ray繞射數據收集與結構解析 25 2.6.1. 蛋白晶體冷凍保護 (cryo-protection) 25 2.6.2. 蛋白晶體之X-ray繞射數據收集 26 三、結果 27 3.0. mAzIN-hAz95-228蛋白複合體 27 3.1. mAzIN-hAz95-228蛋白複合體之大量表現 27 3.2. mAzIN-hAz95-228蛋白複合體之純化 28 3.2.1. 鎳離子親和性層析 (nickel-chelating affinity chromatography) 28 3.2.2. 陰離子交換層析 (anionic exchange chromatography) 28 3.2.3. 膠體過濾層析 (gel filtration chromatography, GFC) 29 3.3. 蛋白質晶體培養 29 3.3.1. 蛋白結晶之條件初步篩選 30 3.3.2. 蛋白結晶之條件微調 30 3.4. 蛋白質結構解析 32 3.4.1. mAzIN-hAz95-228蛋白複合體之X-ray繞射圖譜 32 四、討論 33 4.1. 蛋白質表現 33 4.2. 蛋白質純化 33 4.3. 蛋白質晶體培養 34 圖 37 表 56 參考文獻 64 | |
dc.language.iso | zh-TW | |
dc.title | 哺乳動物抗酶與抗酶抑制蛋白複合體之結構研究 | zh_TW |
dc.title | Structural studies of mammalian antizyme
in complex with antizyme inhibitor | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 曾秀如,徐駿森 | |
dc.subject.keyword | 抗?,抗?抑制因子, | zh_TW |
dc.subject.keyword | antizyme,antizyme inhibitor, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201704151 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-21 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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