人類抗酶與抗酶抑制蛋白複合體之結構研究

Structural studies of human antizyme isoforms in complex with its inhibitor

歐陽陞(Sheng Ou Yang)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201610072

摘要

中文摘要
多胺類化合物(polyamines)為一群在生物體內帶有二個以上正電荷的有機分子,因著帶多價正電的性質,此類物質得以藉由靜電作用力與帶負電之核酸、去氧核醣核酸、脂質、或是蛋白表面酸性胺基酸分佈較多的區域結合,可影響細胞之生長及凋亡。由於多胺的過量表現與惡性腫瘤生成息息相關;因此其濃度受到嚴密的調控。而影響多胺生合成 的速率決定步驟酵素 Onithine decarboxylase (ODC) 於體內的活性高低,是主導生物體內多胺類含量的關鍵因子。當細胞內的多胺含量過高時,會促進抗酶蛋白 (Antizyme, Az) mRNA 發生＋1 的轉譯框架位移(+1 frameshift),使全長的 Az 得以合成。Az 能與 ODC 形成異質二聚體 (Heterodimer) ,而阻止 ODC 形成具有催化活性的同質二聚體 (Homodimer) , Az 與 ODC 所形成的複合體可以在不需泛素 (Ubiquitin) 參與的情況下,被 26S 蛋白酶體 (26S proteasome) 辨認,並造成 ODC 的降解。另外, Az 也能阻斷細胞對外來多胺的攝取,因此, Az 是多胺生合成的負調控因子。而人類基因體中帶 有三種Az亞型,其中的第一亞型 (Az1) 與第二亞型 (Az2) 在組織中的分布很相似,且具有極高度的序列相似性。 但在 in vitro 的實驗中卻發現僅有 Az1 具備讓 ODC 降解的能力,而 Az2 的功能為暫時性地抑制 ODC 的活性。
除此之外,生物體內尚有抗酶抑制因子 (Antizyme inhibitor; AzIN) 能夠與 Az 競爭結合 ODC 的位置,形成 Az 及 AzIN 的異質二聚體,使ODC的活性回覆。Az 結合並可提高 AzIN 的穩定性,減少了泛素參之 AzIN 的降解,達成ODC的正向調節。最近的研究結果顯示,人類惡性肝癌 HCC (Human Hepatocellular Carcinoma) 中發現了 AzIN 之 mRNA 會經 ADAR1 (Adenosine Deaminase Acting on RNA-1) 自發性地 mRNA 編輯(mRNA editing),導致在 AzIN 胺基酸序列第367的絲胺酸被甘胺酸取代,而這樣的結果,會使得 AzIN 對 Az1的親和力上升,增加了 Az1 及 AzIN 複合體的穩定性，導致細胞中 ODC 及多胺含量上升，進而誘發細胞的癌化。
本實驗室一直以來希望藉結構的角度來觀察 Az 分別與 ODC 和 AzIN
ii 之間交互作用的異同,日前我們已解出了解析度達 2.6 A 的 ODC-Az1 晶體結構,且初步得到 Az1110-228-AzIN 較低解析度 (5.8 A) 的晶體結構,本研究的動機為獲得品質更好的 Az-AzIN 晶體,以更深入的了解 Az1-AzIN 的結構及兩者之間交互作用的細節。因此本研究中所使用的策略為：利用 Az2 與 Az1 的高同源性,及 Az1 -AzINS367G 較高的複合體穩定性;希望能幫助我們得到較高解析度的晶體。
本實驗使用了一系列 Az2 重組蛋白與 AzIN 的表達質體,及一系列 Az1 重組蛋白與 AzINS367G 的表達質體,經表現後藉管柱純化獲得相當純度的蛋白複合體。目前雖然 Az2 系列的重組蛋白與 AzIN 的複合體尚未獲得適當晶體生成的條件。但已可初步地得到 Az1119-228-AzINS367G 複合體的結晶條件。未來將再嘗試著獲得其他複合體的晶體生成條件、及品質更好的晶體,以利後續結構的解析。

關鍵字

人類抗酶

並列摘要

Abstract
Polyamines are multivalent organic polycations ubiquitously present in eukaryotic cells. With their polycationic characteristics, polyamines can bind to proteins and nucleic acids via electrostatic interactions to modulate their structures and functions, in turn affecting cell growth and differentiation.
However, abberant accumulation of polyamines is linked to tumorigenesis, thus the intracellular concentrations of polyamines are tightly regulated. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthesis pathway, can regulate the polyamines abundance in cells. Expression of full-length Az (Antizyme) is increased in response to high cellular polyamines levels through the polyamine-induced translational +1 frameshifting mechanism. Az can block the formation of catalytically active ODC homodimer by forming an Az-ODC heterodimer, which not only inhibits ODC enzymatic activity but also trigger ODC degradation via the 26s proteasome in an ubiquitin-independent manner. Furthermore, Az also inhibits the uptake of extracellular polyamine to reduce the cellular polyamide level. Therefore, Az is a negative regulator of cellular polyamines. In mammals, the Az family is consisted of three members (isoforms 1~3). Among them, isoform 1(Az1) and 2 (Az2) exhibit a similar tissue distribution and high sequence similarity. However, previous studies have shown that only Az1 can efficiently promote ODC degradation in vitro and Az2 most likely functions as a reservoir for transient and reversible suppression of ODC activity.
In contrast, AzIN can effectively replenish ODC activity by competing with ODC for Az1 via the formation Az-AzIN heterodimer, which also prolongs the half-life of iv AzIN by preventing its ubiquitination. A recent study show that an mRNA-editing event mediated by the ADAR1 (Adenosine Deaminase Acting on RNA-1) on AzIN mRNA can promote HCC (Human Hepatocellular Carcinoma) by causing a serine-to-glycine substitution at residue 367 of AzIN. The mutated AzIN (AzINS367G) exhibits a higher affinity toward Az, producing a Az1-AzINS367G protein complex with enhanced stability.
To understand the structural details regarding the formation of Az1-AzIN complex, our laboratory has determined the crystal structure of a truncated Az1 in complex with ODC and obtained a lower resolution 5.8 A crystal structure of Az1110-228-AzIN. The main objective of this work is to obtain crystal structures of Az-AzIN at higher resolution and elucidate the interactions between Az1 and AzIN in atomic detail. Toward this goal, two strategies were proposed: (1) obtaining a higher resolution crystal structure of Az2-AzIN by exploting the high similarity between Az1 and Az2 and (2) using the more stable of Az1-AzINS367G complex for structural analysis.
We have constructed a series of recombinant expression vectors for producing Az2-AzIN and Az1-AzINS367G. The reconstituted complexes were purified by using immobilized metal affinity, ion exchange and gel filtration chromatography for crystallization trials. We have successfully identified a condition by which Az1119-228-AzINS367G can be crystallized. However, like the previous Az1110-228-AzIN crsytals produced previously, these crystals diffract only to low resolution. The attemps for crystallzing Az2-AzIN is not yet successful at this time. We will examine whether the diffraction quality of the Az1119-228-AzINS367G crystals can be further improved and continue to search for new crystallization conditions for Az2-AzIN in the future.

並列關鍵字

human antizyme

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