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標題: 人類抗酶抑制因子與抗酶複合體之結晶與初步X-ray單晶繞射分析
Crystallization and preliminary X-ray crystallographic analysis of the human antizyme inhibitor in complex with antizyme
作者: 李佩螢
Lee, Pei-Ying
關鍵字: polyamine
ornithine decarboxylase
antizyme inhibitor
出版社: 生物化學研究所
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摘要: 多元胺是一類結構中具有多個胺基的脂肪族小分子,其於細胞生長、分化以及凋亡皆扮演重要的角色。細胞中多元胺濃度的變化藉由控制鳥胺酸脫羧酶(ornithine decarboxylase, ODC)的蛋白含量而達成,以穩定細胞內的多元胺水平。當細胞內多胺濃度升高時,抗酶(antizyme, AZ)會經由mRNA之轉譯調控而被合成,能特異性地與ODC單體結合,並藉由與泛素無關的途徑(ubiquitin- independent pathway)使ODC被26S蛋白酶體辨認並降解,以抑制多元胺的合成。此外,抗酶還會與胞膜上特定的運輸蛋白結合,降低細胞對多元胺的攝取,反之,當多元胺濃度偏低時,細胞會以抗酶抑制因子(antizyme inhibitor, AZI)來拮抗AZ的功能,為多元胺合成的正調控蛋白,其序列及分子量和ODC相似,但不具有酵素活性。AZI與AZ之間的親和性極高,因而能夠經由競爭釋放與AZ結合的ODC,並恢復其酵素活性。與ODC不同的是,AZI是經由泛素參與之途徑被26S蛋白酶體降解,與AZ結合後,可抑制其與泛素連接酶(Ubiquitin E3 ligase)之間的交互作用,增加其穩定度。最近研究指出,AZ與AZI之間的拮抗作用,除了可穩定細胞內多元胺的水平外,還可調節中心粒的複製,對細胞的分裂反應極為重要。 本論文的主要目的在於解析AZI與AZ形成之蛋白複合體的晶體結構,希望藉此結構進一步了解其兩者之交互作用,並解釋AZ如何避免AZI與泛素連接酶的結合。目前已在數種結晶條件下得到hAZ-hAZI蛋白複合體的晶體。然而,由於這些晶體內部的排列並非十分規則,因而其X-ray繞射訊號的解析度僅達7Å,初步分析為Hexagonal晶系,空間群為P3,晶胞參數 a = b = 163.11 Å, c = 149.69 Å,α= β= 90°, γ =120°。雖然曾試圖以添加物試驗、改變蛋白溶液試驗、crystal annealing、In situ proteolysis等方法提升晶體繞射度,惟目前尚未成功。未來將繼續嘗試其他可能改善晶體品質的方法。
Polyamines are aliphatic cations which play critical roles during cell proliferation, cell death and differentiation. Intracellular polyamine levels are controlled by regulating the protein level of ornithine decoboxylase (ODC), the first enzyme in the polyamine biosynthesis pathway. Antizyme (AZ) is a negative regulator of cellular ODC level and polyamine transport. The elevation of cellular polyamine concentration stimulates a programmed +1 frameshift during translation of the AZ messenger RNA (mRNA), and allows the expression of functional full-length AZ. AZ binds and inactivates ODC by forming AZ-ODC heterodimer, and promotes an ubiquitin-independent degradation by the 26S proteasome, which consequently reduces polyamine synthesis. Mammalian cells contain another relevant regulatory protein termed antizyme inhibitor (AZI), which is homolous to ODC but lacks decarboxylating activity. AZI has a higher affinity for AZ than does ODC, therefore ODC may released from AZ in the presence of AZI and restores the ODC activity. While AZ promotes protolytic destruction of ODC, the degradation of AZI is ubiquitin-dependent and does not require interaction with AZ. Moreover, AZ binding actually stabilizes AZI by inhibiting its ubiquitination. Besides controlling cellular polyamines homeostasis, it has been shown that AZI may regulate the replication of centriole and participate in cell division. To understand how AZ interacts with AZI and to explain how AZ inhibits the poly-ubiqitination of AZI, this study is aimed to determine the structure of AZ-AZI complex. A number of crystallization conditions have been indentified from which crystals of AZ-AZI complex can be obtained. However, these crystals only diffracted X-ray to ~ 7Å resolution. Preliminary analysis indicated that AZ-AZI crystals belongs to space group P3, with unit-cell parameters a = b = 163.11 Å, c = 149.69 Å, α= β= 90°, γ =120°. Additive screen、crystal annealing、optimal buffer searching and in situ proteolysis had been performed to improve crystal quality. However, a useful crystallization condition for AZ-AZI complex remains to be identified.
其他識別: U0005-1002200915120400
Appears in Collections:生物化學研究所



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