Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24023
標題: 人類鳥胺酸脫羧酶與抗酶複合體之純化與晶體培養
Purification and crystallization of human ornithine decarboxylase in complex with antizyme
作者: 林婉婷
Lin, Wan-Ting
關鍵字: ornithine
鳥胺酸脫羧酶抗酶多胺
ornithine decarboxylase
antizyme
polyamine
putrescine
spermidine
spermine
PLP
decarboxylation
crystallography
protein structure
腐胺
亞精胺
精胺
哆醛
脫羧反應
蛋白結晶
結構
養晶
出版社: 生物化學研究所
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摘要: 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。
Polyamines (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.
URI: http://hdl.handle.net/11455/24023
其他識別: U0005-2907200913492300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2907200913492300
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