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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 | Fang-Yu Li | en |
dc.contributor.author | 黎芳瑜 | zh_TW |
dc.date.accessioned | 2021-06-17T08:35:32Z | - |
dc.date.available | 2020-08-26 | |
dc.date.copyright | 2019-08-26 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-08 | |
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Wallace, H.M., A.V. Fraser, and A. Hughes, A perspective of polyamine metabolism. Biochem J, 2003. 376(Pt 1): p. 1-14. 10. Yamashita, T., et al., Role of polyamines at the G1/S boundary and G2/M phase of the cell cycle. The International Journal of Biochemistry & Cell Biology, 2013. 45(6): p. 1042-1050. 11. Gerner, E.W. and F.L. Meyskens, Jr., Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer, 2004. 4(10): p. 781-92. 12. Pegg, A.E., Polyamine Metabolism and Its Importance in Neoplastic Growth and as a Target for Chemotherapy. 1988. 48(4): p. 759-774. 13. Pegg, A.E., Spermidine/spermine-N1-acetyltransferase: a key metabolic regulator. 2008. 294(6): p. E995-E1010. 14. Igarashi, K., Physiological Functions of Polyamines and Regulation of Polyamine Content in Cells. YAKUGAKU ZASSHI, 2006. 126(7): p. 455- 471. 15. Poulin, R., R.A. Casero, and D. Soulet, Recent advances in the molecular biology of metazoan polyamine transport. Amino acids, 2012. 42(2-3): p. 711- 723. 16. Almrud, J.J., et al., Crystal structure of human ornithine decarboxylase at 2.1 å resolution: structural insights to antizyme binding11Edited by I. A. Wilson. Journal of Molecular Biology, 2000. 295(1): p. 7-16. 17. Pegg, A.E., Regulation of ornithine decarboxylase. J Biol Chem, 2006. 281(21): p. 14529-32. 18. Dunathan, H.C., Conformation and reaction specificity in pyridoxal phosphate enzymes. Proceedings of the National Academy of Sciences of the United States of America, 1966. 55(4): p. 712-716. 19. Jackson, L.K., et al., Altering the Reaction Specificity of Eukaryotic Ornithine Decarboxylase. Biochemistry, 2000. 39(37): p. 11247-11257. 20. Shantz, L.M. and A.E. Pegg, Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway. The International Journal of Biochemistry & Cell Biology, 1999. 31(1): p. 107-122. 21. Murakami, Y., et al., Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature, 1992. 360(6404): p. 597-599. 22. Moshier, J.A., et al., Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells. Nucleic Acids Res, 1992. 20(10): p. 2581-90. 23. Lan, L., C. Trempus, and S.K. Gilmour, Inhibition of Ornithine Decarboxylase (ODC) Decreases Tumor Vascularization and Reverses Spontaneous Tumors in ODC/Ras Transgenic Mice. 2000. 60(20): p. 5696- 5703. 24. 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, 2013. 133(6): p. 1323-33. 25. Heller, J.S., W.F. Fong, and E.S. Canellakis, Induction of a protein inhibitor to ornithine decarboxylase by the end products of its reaction. 1976. 73(6): p. 1858-1862. 26. Kahana, C., Antizyme and antizyme inhibitor, a regulatory tango. Cell Mol Life Sci, 2009. 66(15): p. 2479-88. 27. Coffino, P., Antizyme, a mediator of ubiquitin-independent proteasomal degradation. Biochimie, 2001. 83(3-4): p. 319-23. 28. Newman, R.M., et al., Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression. J Biol Chem, 2004. 279(40): p. 41504- 11. 29. Lim, S.K. and G. Gopalan, Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A. Oncogene, 2007. 26(46): p. 6593-6603. 30. Gruendler, C., et al., Proteasomal Degradation of Smad1 Induced by Bone Morphogenetic Proteins. 2001. 276(49): p. 46533-46543. 31. Kasbek, C., C.-H. Yang, and H.A. Fisk, Antizyme restrains centrosome amplification by regulating the accumulation of Mps1 at centrosomes. Molecular biology of the cell, 2010. 21(22): p. 3878-3889. 32. 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, 2016. 471(4): p. 646-51. 33. Hoffman, D.W., et al., Solution Structure of a Conserved Domain of Antizyme: A Protein Regulator of Polyamines. Biochemistry, 2005. 44(35): p. 11777-11785. 34. Rom, E. and C. Kahana, Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(9): p. 3959-3963. 35. Matsufuji, S., et al., Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell, 1995. 80(1): p. 51-60. 36. Kurian, L., et al., Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature, 2011. 477(7365): p. 490-4. 37. Baranov, P.V., et al., RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression. Nucleic acids research, 2001. 29(1): p. 264-267. 38. Wu, H.Y., et al., Structural basis of antizyme-mediated regulation of polyamine homeostasis. Proc Natl Acad Sci U S A, 2015. 112(36): p. 11229- 34. 39. Hsieh, J.Y., et al., Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor. PLoS One, 2011. 6(9): p. e24366. 40. Zhang, M., C.M. Pickart, and P. Coffino, Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate. The EMBO journal, 2003. 22(7): p. 1488-1496. 41. Fujita, K., Y. Murakami, and S. Hayashi, A macromolecular inhibitor of the antizyme to ornithine decarboxylase. 1982. 204(3): p. 647-652. 42. Murakami, Y., et al., Cloning of Antizyme Inhibitor, a Highly Homologous Protein to Ornithine Decarboxylase. 1996. 271(7): p. 3340-3342. 43. Kim, S.W., et al., Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism. 2006. 119(12): p. 2583- 2591. 44. Mangold, U., et al., Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification. Oncogene, 2008. 27(5): p. 604-13. 45. Qiu, S., J. Liu, and F. Xing, Antizyme inhibitor 1: a potential carcinogenic molecule. Cancer Sci, 2017. 108(2): p. 163-169. 46. Chen, L., et al., Recoding RNA editing of AZIN1 predisposes to hepatocellular carcinoma. Nat Med, 2013. 19(2): p. 209-16. 47. Albeck, S., et al., Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function. Protein Sci, 2008. 17(5): p. 793-802. 48. Russo Krauss, I., et al., Increasing the X-ray diffraction power of protein crystals by dehydration: the case of bovine serum albumin and a survey of literature data. International journal of molecular sciences, 2012. 13(3): p. 3782-3800. 49. McMahon, C., et al., Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nature structural & molecular biology, 2018. 25(3): p. 289-296. 50. Soda, K., Polyamine Metabolism and Gene Methylation in Conjunction with One-Carbon Metabolism. Int J Mol Sci, 2018. 19(10). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74433 | - |
dc.description.abstract | 多胺 (polyamine) 是一類對細胞存活而言不可或缺的重要小分子,其帶有 多價正電荷,能透過靜電力 (electrostatic interaction) 與帶負電的分子進行交互 作用,例如:DNA、RNA 或膜蛋白等,因此廣泛的參與在許多細胞生理作用, 例如:轉錄、轉譯及訊息傳遞等,進而影響細胞的生長與分化。已知多胺在細 胞中的濃度會隨著細胞週期而變化,以維持細胞的生理功能,然而許多研究都 發現若多胺濃度異常升高,會造成細胞不正常生長與增生,導致腫瘤的形成, 所以細胞中多胺的濃度必須嚴密調控在一定的範圍內。
細胞中的多胺可以藉由細胞外攝入,也可以在胞內直接生合成,在合成的 路徑中鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 所催化的反應,是合成多 胺的第一步驟,也是整個生合成途徑的速率決定步驟,因此 ODC 在調控多胺 的濃度中扮演一個相當重要的角色,透過控制 ODC 的含量,可以調控細胞中 多胺的濃度。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,需要一個名為抗酶 (antizyme, Az) 的 調控蛋白參與,Az 單體能夠透過競爭的方式與 ODC 單體形成異質二聚體 (heterodimer),使具有酵素活性的 ODC 雙體無法形成,藉此抑制 ODC 活性, 且 Az-ODC 異質二聚體會被 26S 蛋白酶體所辨認,並進一步將 ODC 降解。 除此之外,Az 也會調控細胞膜上的多胺搬運蛋白 (polyamine transporter) ,減 少對外源性多胺的攝入;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蛋白,其與 Az 具有拮抗的功能,AzIN 會與 ODC 競爭和 Az 的結合, 抑制 Az 的作用,藉此提升細胞中多胺的濃度,因 此,透過 Az 與 AzIN 對 ODC 的調控,能使細胞中多胺的濃度維持在適當範圍。 在 2015 年本實驗室解出 Az-ODC 與 Az-AzIN 複合體的晶體結構,對於 Az 調控 ODC 的結構基礎提供了進一步的了解,然而受限於此 Az-AzIN 複合 體結構解析度較低的緣故 (~5.8 Å) ,對於 Az 及 AzIN 之間的交互作用細節依舊不了解。本研究的目標為製備品質更好的晶體,得到 Az-AzIN 複合體更 高解析度的結構,以了解二者交互作用細節,研究策略上企圖利用與人類 Az (hAz) 及人類 AzIN (hAzIN) 序列高度相似的小鼠 Az (mAz) 及小鼠 AzIN (mAzIN) ,以更換物種的方式,或基於人類與小鼠蛋白結構與功能具高度保留 性、因此可能形成跨物種蛋白複合體的假設,期待得到不同的晶體,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,在未來我們將嘗試利用不同的蛋白晶體冷凍保護方法改善 這些晶體的繞射情形,也持續篩選新的養晶條件。 | zh_TW |
dc.description.abstract | Polyamines are multivalent cations that are involved in a large number of cellular processes, ranging from modulation of cell growth to regulation of differentiation. Given its key role in promoting cell proliferation, the cellular concentrations of polyamines are tightly regulated, and elevated polyamine level has been found to associate with numerous types of neoplastic transformations. Thus, inhibiting polyamine production may be an effective approach for treating cancer.
The biosynthesis of polyamines starts from the reaction catalyzed by ornithine decarboxylase (ODC) which also serves as the rate-limiting enzyme of this pathway. ODC is enzymatically active only when exists as a homodimer, and the negative regulation of its activity by the regulatory protein antizyme (Az) is achieved via disrupting the ODC homodimer. Az can decrease polyamine levels through binding and converting ODC into an enzymatically inactive Az-ODC heterodimer. In addition, the Az-ODC heterodimer can be recognized by the 26S proteasome, which targets ODC for an ubiquitin-independent degradation. Besides interacting with ODC, Az also suppresses polyamine uptake from the extracellular environment through inhibiting polyamine transporters. Thus, an increase in Az lowers the levels of ODC and polyamine, in turn reducing cell growth. On the other hand, the expression of a catalytically dead ODC homolog named antizyme inhibitor (AzIN) increases polyamine levels by competing with ODC for Az to prevent Az-mediated ODC degradation. Together ODC, Az, and AzIN form a delicate regulatory circuit to coordinate intracellular polyamine homeostasis.Though the overall three-dimensional structures of Az-AzIN and Az-ODC complexes are similar, Az-AzIN is more stable than Az-ODC and only the latter can be recognized by the 26S proteasome. It has been suggested that small molecule- mediated interruption of Az-AzIN heterodimerization without affecting the stability of Az-ODC heterodimer may be exploited in anticancer therapy by blocking polyamine production. But our understanding of the structural differences between Az-AzIN and Az-ODC complexes have remained incomplete due to the lack of detailed structural information about the Az-AzIN complexes. Thus, the goal of my thesis research is to determine a crystal structure of Az-AzIN at high resolution. Since previously obtained crystals formed by human AzIN (hAzIN) and an N- terminal truncated form of human Az (hAz95-228) diffract only to low resolution, we tested whether crystals of the cross-species complexes formed by mouse Az (mAz) with either mouse AzIN (mAzIN) or hhAzIN, and hAz with mAzIN may produce better diffraction data. We have successfully established a protein expression system for producing large amount of mAzIN-hAz95-228 complex and used immobilized metal affinity, ion exchange and gel filtration chromatography for purification. Using vapor diffusion crystallization technique, various conditions for growing mAzIN- hAz95-228 crystals have been identified. However, only low resolution diffraction data were obtained from these crystals. Different post-crystallization treatments, including crystal dehydration and cryo-protection procedures, will be employed to improve the diffraction quality of these crystals. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:35:32Z (GMT). No. of bitstreams: 1 ntu-108-R06442018-1.pdf: 4217749 bytes, checksum: d03355cb6f45e197e7049e78ff0c22a6 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 謝誌 ................................................................................................ I
摘要 ............................................................................................... II Abstract....................................................................................... IV 縮寫表 .......................................................................................... VI 目錄 ............................................................................................ VII 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. 多胺之合成代謝路徑 ...............................................................1 1.1.3. 胞內多胺含量之調控 ...............................................................2 1.2. 鳥胺酸脫羧酶 (ornithine decarboxylase, ODC)..........................3 1.2.1. 鳥胺酸脫羧酶之結構 ...............................................................3 1.2.2. 鳥胺酸脫羧酶之催化機制 ........................................................3 1.2.3. 鳥胺酸脫羧酶與疾病 ..............................................................4 1.3. 抗酶 (antizyme, Az) .................................................................5 1.3.1. 抗酶之生理功能與重要性 ........................................................5 1.3.2. 抗酶之結構 ............................................................................5 1.3.3. 抗酶之調控 ............................................................................6 1.3.4. 抗酶與鳥胺酸脫羧酶之交互作用與降解機制..............................6 1.4. 抗酶抑制因子 (antizyme inhibitor, AzIN)...................................7 1.4.1. 抗酶抑制因子生理功能與重要性...............................................7 1.4.2. 抗酶抑制因子與疾病 ..............................................................8 1.4.3. 抗酶抑制因子之結構 ..............................................................8 1.4.4. 抗酶抑制因子與抗酶之交互作用..............................................8 1.5. 研究目的...................................................................................9 二、 材料與方法 ............................................................................10 2.1. 蛋白質表現系統.......................................................................10 2.1.1. 蛋白質體建構........................................................................10 2.1.2. 表現蛋白菌株........................................................................10 2.1.3. 製備勝任細胞 (competent cell).............................................10 2.1.4. 轉型作用 (transformation)....................................................11 2.1.5. 凍菌保存 (stock)...................................................................11 2.2. 蛋白小量表現量測試.................................................................11 2.2.1. Az-AzIN 蛋白複合體之共表達................................................12 2.2.2. 蛋白表現測試........................................................................12 2.2.3. 膠體電泳 (Gel electrophoresis) 分析....................................12 2.3 蛋白質純化...............................................................................14 2.3.1. 蛋白之大量表現.....................................................................14 2.3.2. 破菌與蛋白萃取....................................................................14 2.3.3. 液相層析 (liquid chromatography).......................................15 2.4. 蛋白質濃縮及定量 ...................................................................17 2.4.1. 蛋白複合體濃縮.....................................................................17 2.4.2. 蛋白複合體定量....................................................................17 2.5. 蛋白質晶體培養.......................................................................18 2.5.1. 緩衝溶液篩選 (buffer screen) ..............................................18 2.5.2. PCT (Pre-Crystallization Test).............................................18 2.5.3. 蛋白結晶之晶體培養方法 ......................................................19 2.5.4. 蛋白結晶條件初步篩選 .........................................................19 2.5.5. 蛋白結晶條件微調................................................................20 2.5.6. 晶體脫水 (Crystal dehydration)...........................................20 2.6. 蛋白質晶體之 X-ray 繞射數據解析 ...........................................21 2.6.1. 蛋白晶體冷凍保護 (cryo-protection).....................................21 2.6.2. 蛋白晶體之 X-ray 繞射數據收集 ............................................21 2.7. 奈米抗體篩選 (Nanobody selection)........................................21 2.7.1. 酵母表面展示系統(yeast surface display)表現奈米抗體庫(library) ..........22 2.7.1. 蛋白樣品準備.......................................................................23 2.7.2. Nanobody 篩選...................................................................24 2.7.3. Nanobody 之鑑定與表現質體建構 ........................................25 三、 結果 ......................................................................................27 3.1. Az-AzIN 蛋白複合體 ...............................................................27 3.2. 蛋白複合體之表現 ..................................................................27 3.3. 蛋白複合體之純化 ..................................................................28 3.3.1. 鎳離子親和性管柱 (nickel-chelating affinity chromatography).................28 3.3.2. 陰離子交換層析 (anionic exchange chromatography)..........29 3.3.3. 膠體過濾層析 (gel filtration chromatography, GFC).............29 3.4. 蛋白質晶體培養......................................................................30 3.4.1. 緩衝溶液篩選 (buffer screen) ..............................................30 3.4.2. 蛋白結晶條件初步篩選..........................................................31 3.4.3. 蛋白結晶條件微調 ...............................................................32 3. 4.4. 晶體脫水 (Crystal dehydration)...........................................33 3.5. 蛋白質結構解析.......................................................................33 四、 討論 ......................................................................................35 4.1. 蛋白質表現..............................................................................35 4.2. 蛋白質純化.............................................................................35 4.3. 蛋白質晶體培養......................................................................36 圖..................................................................................................38 表..................................................................................................64 參考文獻 .......................................................................................75 附錄 .............................................................................................79 | |
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 | Toward high-resolution structural studies of the mammalian antizyme complexed with antizyme inhibitor | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐駿森,曾秀如 | |
dc.subject.keyword | 多胺,鳥胺酸脫羧?,抗?,抗?抑制因子, | zh_TW |
dc.subject.keyword | polyamine,ornithine decarboxylase,antizyme,antizyme inhibitor, | en |
dc.relation.page | 79 | |
dc.identifier.doi | 10.6342/NTU201902866 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-12 | |
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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