Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24023
DC FieldValueLanguage
dc.contributor劉光耀zh_TW
dc.contributor洪慧芝zh_TW
dc.contributor.advisor詹迺立zh_TW
dc.contributor.author林婉婷zh_TW
dc.contributor.authorLin, Wan-Tingen_US
dc.contributor.other中興大學zh_TW
dc.date2010zh_TW
dc.date.accessioned2014-06-06T07:21:49Z-
dc.date.available2014-06-06T07:21:49Z-
dc.identifierU0005-2907200913492300zh_TW
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Atkins, (2000) Discovery of a spermatogenesis stage-specific ornithine decarboxylase antizyme: antizyme 3. Proc Natl Acad Sci U S A 97: 4808-4813. Jackson, L. K., H. B. Brooks, A. L. Osterman, E. J. Goldsmith & M. A. Phillips, (2000) Altering the reaction specificity of eukaryotic ornithine decarboxylase. Biochemistry 39: 11247-11257. Kern, A. D., M. A. Oliveira, P. Coffino & M. L. Hackert, (1999) Structure of mammalian ornithine decarboxylase at 1.6 A resolution: stereochemical implications of PLP-dependent amino acid decarboxylases. Structure 7: 567-581. Li, X. & P. Coffino, (1993) Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein. Mol Cell Biol 13: 2377-2383. Li, X. & P. Coffino, (1994) Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase. Mol Cell Biol 14: 87-92. Mangold, U., (2006) Antizyme inhibitor: mysterious modulator of cell proliferation. 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dc.identifier.urihttp://hdl.handle.net/11455/24023-
dc.description.abstract多元胺 (polyamine) 在細胞中是帶多個正電荷的一級胺, 包含腐胺 (putrescine)、亞精胺 (spermidine) 及精胺 (spermine),其能與帶負電荷的 DNA、RNA 或蛋白質交互作用,對細胞的生長(proliferation)、分化 (differentiation) 及凋亡 (apoptosis) 極為重要。由於過高濃度的多元胺會導致細胞癌化,因此多元胺在胞內的濃度受到嚴密的調控。人類鳥胺酸脫羧酶 (Ornithine decarboxylase,ODC; EC 4.1.1.17) 能夠催化鳥胺酸 (ornithine),使其脫去一個羧基而得到腐胺(putrescine),此反應為多元胺合成途徑的起始與速率決定 (rate-limiting) 步驟,亦是多元胺合成主要的調控點。在高濃度多元胺的情況下,胞內會藉由 mRNA之轉譯調控而合成抗酶 (Antizyme, AZ ),AZ 能夠與 ODC 單體結合以抑制其催 化活性並促進其降解,進而降低多元胺在細胞中的濃度。另外,多元胺的濃度調節亦受到抗酶抑制因子 (Antizyme inhibitor, AZI ) 的調控,AZI 與 ODC 之胺基酸序列及分子量相似,但不具催化活性。由於 AZI 與 AZ 之親合力遠高於ODC,因此能和 AZ 結合並抑制 ODC-AZ 之交互作用,使 AZ 釋放 ODC 以恢復其催化活性,進而提升多元胺在細胞中的濃度。 人類 ODC 由 461 個胺基酸組成,分子量約 53 kDa,兩個 ODC 單體 (monomer) 間係以頭尾相連 (head-to-tail) 的方式組成雙聚體 (homodimer),並以其活性區 (active site) Lys69 以 Schiff-base 與輔酶5’-磷酸吡哆醛(pyridoxal 5’-phosphate, PLP)結合,形成一個具有催化活性的酵素。AZ 結合於 ODC 胺基酸序列之 117-140 區域,使 ODC 由 homodimer 狀態轉變成 ODC-AZ heterodimer,此時失去催化活性的 ODC 裸露出 C 端 degradation signal,引導其為 proteasome 所辨識並被降解。 本實驗目的希望以蛋白質結構分析的角度來探討 AZ 與 ODC 間的交互作用,進而解釋 AZ 辨認並促使 ODC 降解的分子機制。雖然全長 ODC 與 AZ N-端刪除突變 (AZ110) 所形成的複合體無法形成晶體,但在將其結構中之不穩定區域刪除而得的 ODC(@299-310) 可和 AZ110 形成穩定的複合體,此複合體可在2% PEG 400, 0.1 M Na HEPES pH7.5 , 2.0 M Ammonium Sulfate 條件下形成四角柱狀晶體,初步 X-ray 繞射分析結果發現此晶體確實為蛋白晶體,日後將繼續改善結晶條件並且尋找適合之冷凍保護劑 (cryo protection buffer) 及冷凍保護方式,以利後續之結構解析。zh_TW
dc.description.abstractPolyamines (putrescine, spermidine, and spermine) are primary amines which exist as polycations under physiological pH conditions. These compounds are required for cell growth, differentiation, and apoptosis by participating in many cellular processes, including chromosome condensation, maintenance of DNA structure, RNA processing, translation and protein activation. Overexpression of polyamne is known to induce cell transformation and tumorigensis. Ornithine decarboxylase (ODC; EC 4.1.1.17) performs the first and rate-determining step in polyamine biosynthesis, in which ornithine is decarboxylated to from putrescine. Elevated cellular polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme (AZ) mRNA, resulting in subsequent binding of AZ to ODC monomer and AZ-dependent targeting of ODC for proteasomal degradation. Antizyme inhibitor (AZI) also regulates cellular polyamine homeostasis by binding to AZ. AZI is highly homologous to ODC but is not enzymatically active. Because AZI binds to AZ with much higher affinity than ODC,AZI can upregulate polyamine biosynthesis by suppressing the interaction between ODC and AZ. Human ODC is a 53 kDa pyridoxal 5'-phosphate (PLP)-dependent enzyme consists of 461 amino acids. The active form of ODC is a head-to-tail homodimer,and the active site residue Lys69 forms a Schiff-base linkage with PLP. Residues 117-140 of ODC are critical for its association with AZ. AZ binding disrupts ODC homodimer and presumably induces a conformational change on ODC, resulting in the exposure of the C-terminal degradation signal which allows proteolysis of ODC by the 26S proteasome to occur. In this study, we try to understand how AZ recognizes ODC and how the proteasomal degradation of ODC is promoted by AZ binding by determining the crystal structure of AZ-ODC complex. Although the complex formed by full-length ODC and N-terminal truncated AZ (AZ110) failed to be crystallized, we generated a mutant ODC (ODC(@299-310)) in which the flexible surface loop spanning residues 299 to 310 was deleted. We show that the ODC(@299-310) can associate with AZ110 to form heterodimer, and the resulting complex has been successfully crystallized. We are now fine-tuning the conditions for crystallization and cryo-protection to facilitate structure determination.en_US
dc.description.tableofcontents中文摘要---------------------------------------------------I Abstract-------------------------------------------------III 一、緒論---------------------------------------------------1 1-1 多胺 (polyamine) 在生理上的意義------------------------1 1-2 多胺的代謝途徑-----------------------------------------1 1-3 鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 的功能與結構---------------------------------------------------------2 1-4 ODC 的催化反應-----------------------------------------3 1-5 ODC 的降解調控-----------------------------------------4 二、 實驗方法----------------------------------------------8 2-1 製備勝任細胞 (competent cell)--------------------------8 2-2 轉型作用 (transformation)------------------------------8 2-3 質體製備-----------------------------------------------8 2-4 質體構築 (plasmid construct)---------------------------9 2-5 ODC-AZ110 蛋白複合體之共表達--------------------------12 2-6 ODC-AZ110 共表達之蛋白複合體的純化--------------------14 2-7 晶體培養----------------------------------------------19 2-8 胰蛋白酶 (trypsin) 部分水解 (limited proteolysis )----20 2-9 西方墨點法 (Western blot)-----------------------------21 三、 結果-------------------------------------------------22 3-1 ODC-AZ110 蛋白複合體的表現、純化與晶體培養------------22 3-2 ODC-AZ110 蛋白複合體之胰蛋白酶 (trypsin) 部分水解(limited proteolysis) 與分子篩管柱層析純化----------------24 3-3 ODC()299-310)-AZ110 複合體的表現與純化與晶體培養------23 四、討論--------------------------------------------------29 4-1 ODC-AZ119、ODC()299-310)–AZ119 的純化----------------30 4-2 胰蛋白酶 (trypsin) 部分水解---------------------------30 4-3 ODC()299-310)-AZ110 蛋白複合體之晶體培養--------------31 4-4 醫學應用與展望----------------------------------------32 參考文獻--------------------------------------------------33 圖表------------------------------------------------------36 附錄------------------------------------------------------72zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2907200913492300en_US
dc.subjectornithineen_US
dc.subject鳥胺酸脫羧酶抗酶多胺zh_TW
dc.subjectornithine decarboxylaseen_US
dc.subjectantizymeen_US
dc.subjectpolyamineen_US
dc.subjectputrescineen_US
dc.subjectspermidineen_US
dc.subjectspermineen_US
dc.subjectPLPen_US
dc.subjectdecarboxylationen_US
dc.subjectcrystallographyen_US
dc.subjectprotein structureen_US
dc.subject腐胺zh_TW
dc.subject亞精胺zh_TW
dc.subject精胺zh_TW
dc.subject哆醛zh_TW
dc.subject脫羧反應zh_TW
dc.subject蛋白結晶zh_TW
dc.subject結構zh_TW
dc.subject養晶zh_TW
dc.title人類鳥胺酸脫羧酶與抗酶複合體之純化與晶體培養zh_TW
dc.titlePurification and crystallization of human ornithine decarboxylase in complex with antizymeen_US
dc.typeThesis and Dissertationzh_TW
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