哺乳動物抗酶與抗酶抑制蛋白複合體之結構研究

Structural studies of mammalian antizyme in complex with antizyme inhibitor

莊子瑩(Tzu-Ying Chuang)

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(2017年)

https://doi.org/10.6342/NTU201704151

摘要

多胺 (polyamine) 是一類帶有多價正電性的小分子脂肪族化合物，廣泛地存在於自然界中，無論原核及真核細胞中都能觀察到多胺的存在。多胺的多價正電性使其能夠與一些帶負電分子，例如DNA、RNA和磷脂等，或是與蛋白質表面帶有負電性的區域產生可逆的靜電交互作用 (electrostatic interaction) ，進而能參與在生物體中許多重要的生理功能調控，例如：細胞生長、增殖及調節細胞分化等。多胺廣泛地影響了細胞的生長、生存與凋亡，許多研究都證實了多胺濃度異常與正常細胞癌化具有密切相關性，也因此，多胺於細胞中的濃度必須受到嚴密的調控，保持在一定範圍內的恆定，才能使細胞維持正常的生理功能。
在多胺的生合成路徑中，鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 是催化多胺合成的第一步驟，也是整個生合成反應中的速率決定步驟，其在調控多胺於胞內的濃度上，扮演一個相當重要的角色。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，需要一特別調控蛋白的參與，名為抗酶 (antizyme, Az)，Az的單體能夠透過競爭的方式與ODC單體形成異質二聚體 (heterodimer)，抑制ODC活性，並進一步使其被26S 蛋白酶體所降解；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調控蛋白，其與Az具有截然不同的功能，AzIN會與ODC競爭和Az的結合，抑制Az的作用，藉由Az與AzIN相互拮抗，能使整個調控機制的運作更加精準。
過去我們實驗室已解出 Az-ODC的複合體結構，使我們對於Az調控ODC的機制有一定程度的了解，然而受限於先前所發表的Az-AzIN複合體結構解析度不足的緣故 (~5.8 Å) ，我們對於Az及AzIN之間的交互作用細節依舊不甚了解。本研究的目標即在於得到品質更好的晶體、解析出Az-AzIN複合體更精確的結構，以了解二者交互作用細節，就策略而言本研究企圖以跨物種間蛋白結構與功能具高度保留性的概念來突破晶體解析度不佳的困境。
我們實驗室過去的研究發現，人類AzIN (hAzIN) 與Az進行交互作用的界面胺基酸與小鼠AzIN (mAzIN) 的胺基酸有高度保留性及相似性，因此我們期待透過得到小鼠AzIN與人類Az (hAz) 所形成的晶體，藉此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，在未來我們將嘗試改善這些晶體的繞射情形，利用不同的蛋白晶體冷凍保護方法，也持續篩選新的養晶條件。

關鍵字

抗酶 ； 抗酶抑制因子

並列摘要

Polyamines are small multivalent organic polycations ubiquitously present in eukaryotic cells. These compounds play multiple regulatory roles in mammalian physiology owing to their polycationic characteristics. For example, polyamines can bind to proteins and nucleic acids via electrostatic interactions and modulate their structure and functions, in turn affecting many cellular processes, including cell growth and differentiation. It has been shown that intracellular polyamine homeostasis is important for cells to maintain normal physiological functions. Aberrant accumulation of polyamine is associated with cell transformation and tumorigenesis. Therefore, intracellular polyamine level must be under tight control.
Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the rate-limiting step in the polyamine biosynthesis pathway. The activity and stability of this homodimeric protein is controlled by the regulatory protein antizyme (Az). Abundance of full-length Az is increased in response to high intracellular polyamine level through the polyamine-induced translational +1 frameshifting during the translation of Az mRNA. Az can disrupt the formation of active ODC homodimers by forming a tight 1:1 binary complex with ODC monomer, which not only inhibits catalytic activity of ODC, but triggers ODC degradation via the 26S proteasome in an ubiquitin-independent manner. Furthermore, Az can also inhibit polyamine uptake into cells, which reduce the intracellular polyamine level. Therefore, Az is regarded as a negative regulator of cellular polyamines.
Antizyme inhibitor (AzIN) is another major regulatory protein involved in maintaining polyamine homeostasis. In contrast to Az, AzIN functions as a positive regulator of polyamine levels. AzIN competes with ODC for binding to Az. The formation of Az-AzIN heterodimer leads to the release of ODC and effectively replenishes ODC activity. Thus, both AZ and AzIN are important parts of the auto-regulatory circuits designed to maintain optimal levels of polyamine within a cell.
To understand the structural details regarding the formation of Az-AzIN complex, our laboratory has determined the crystal structure of a truncated Az95-228 in complex with ODC and obtained a lower resolution 5.8 Å crystal structure of Az110-228-AzIN. The major objective of this work is to obtain a crystal structure of Az-AzIN at higher resolution and elucidate the interactions between Az and AzIN in atomic detail.
To achieve this goal, we tested whether human Az (hAz) can complex with mouse AzIN (mAzIN), and whether the resultant complex can be crystallized for structural characterization. According to the previous studies of our laboratory, we have found that the interface residues of human and mouse AzIN are highly conserved.
We have successfully built a protein expression and purification system that can be used to produce large amount of highly purified hAZ-mAzIN complex. Specifically, purification was conducted using immobilized metal affinity, ion exchange and gel filtration chromatography. Moreover, we have successfully identified several conditions by which hAz95-228-mAzIN can be crystallized. However, these crystals diffract only to low resolution at the moment. We will examine whether the diffraction quality of hAz95-228-mAzIN crystals can be further improved. Also, we will continue to search for new crystallization conditions in the future.

並列關鍵字

antizyme ； antizyme inhibitor

參考文獻

1 Miller-Fleming, L., Olin-Sandoval, V., Campbell, K. & Ralser, M. Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell. J Mol Biol 427, 3389-3406, doi:10.1016/j.jmb.2015.06.020 (2015).

2 Minois, N., Carmona-Gutierrez, D. & Madeo, F. Polyamines in aging and disease. Aging (Albany NY) 3, 716-732, doi:10.18632/aging.100361 (2011).

3 Bachrach, U. The early history of polyamine research. Plant Physiol Biochem 48, 490-495, doi:10.1016/j.plaphy.2010.02.003 (2010).

4 Iacomino, G., Picariello, G. & D'Agostino, L. DNA and nuclear aggregates of polyamines. Biochim Biophys Acta 1823, 1745-1755, doi:10.1016/j.bbamcr.2012.05.033 (2012).

5 Lightfoot, H. L. & Hall, J. Endogenous polyamine function--the RNA perspective. Nucleic Acids Res 42, 11275-11290, doi:10.1093/nar/gku837 (2014).