哺乳動物抗酶與抗酶抑制蛋白複合體之結構研究

Toward high-resolution structural studies of the mammalian antizyme complexed with antizyme inhibitor

黎芳瑜(Fang-Yu Li)

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(2019年)

https://doi.org/10.6342/NTU201902866

摘要

多胺 (polyamine) 是一類對細胞存活而言不可或缺的重要小分子，其帶有 多價正電荷，能透過靜電力 (electrostatic interaction) 與帶負電的分子進行交互 作用，例如:DNA、RNA 或膜蛋白等，因此廣泛的參與在許多細胞生理作用， 例如:轉錄、轉譯及訊息傳遞等，進而影響細胞的生長與分化。已知多胺在細 胞中的濃度會隨著細胞週期而變化，以維持細胞的生理功能，然而許多研究都 發現若多胺濃度異常升高，會造成細胞不正常生長與增生，導致腫瘤的形成， 所以細胞中多胺的濃度必須嚴密調控在一定的範圍內。
細胞中的多胺可以藉由細胞外攝入，也可以在胞內直接生合成，在合成的 路徑中鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 所催化的反應，是合成多 胺的第一步驟，也是整個生合成途徑的速率決定步驟，因此 ODC 在調控多胺 的濃度中扮演一個相當重要的角色，透過控制 ODC 的含量，可以調控細胞中 多胺的濃度。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，需要一個名為抗酶 (antizyme, Az) 的 調控蛋白參與，Az 單體能夠透過競爭的方式與 ODC 單體形成異質二聚體 (heterodimer)，使具有酵素活性的 ODC 雙體無法形成，藉此抑制 ODC 活性， 且 Az-ODC 異質二聚體會被 26S 蛋白酶體所辨認，並進一步將 ODC 降解。 除此之外，Az 也會調控細胞膜上的多胺搬運蛋白 (polyamine transporter) ，減 少對外源性多胺的攝入;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蛋白，其與 Az 具有拮抗的功能，AzIN 會與 ODC 競爭和 Az 的結合， 抑制 Az 的作用，藉此提升細胞中多胺的濃度，因 此，透過 Az 與 AzIN 對 ODC 的調控，能使細胞中多胺的濃度維持在適當範圍。
在 2015 年本實驗室解出 Az-ODC 與 Az-AzIN 複合體的晶體結構，對於 Az 調控 ODC 的結構基礎提供了進一步的了解，然而受限於此 Az-AzIN 複合 體結構解析度較低的緣故 (~5.8 Å) ，對於 Az 及 AzIN 之間的交互作用細節依舊不了解。本研究的目標為製備品質更好的晶體，得到 Az-AzIN 複合體更 高解析度的結構，以了解二者交互作用細節，研究策略上企圖利用與人類 Az (hAz) 及人類 AzIN (hAzIN) 序列高度相似的小鼠 Az (mAz) 及小鼠 AzIN (mAzIN) ，以更換物種的方式，或基於人類與小鼠蛋白結構與功能具高度保留 性、因此可能形成跨物種蛋白複合體的假設，期待得到不同的晶體，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，在未來我們將嘗試利用不同的蛋白晶體冷凍保護方法改善 這些晶體的繞射情形，也持續篩選新的養晶條件。

關鍵字

多胺 ； 鳥胺酸脫羧酶 ； 抗酶 ； 抗酶抑制因子

並列摘要

Polyamines are multivalent cations that are involved in a large number of cellular processes, ranging from modulation of cell growth to regulation of differentiation. Given its key role in promoting cell proliferation, the cellular concentrations of polyamines are tightly regulated, and elevated polyamine level has been found to associate with numerous types of neoplastic transformations. Thus, inhibiting polyamine production may be an effective approach for treating cancer.
The biosynthesis of polyamines starts from the reaction catalyzed by ornithine decarboxylase (ODC) which also serves as the rate-limiting enzyme of this pathway. ODC is enzymatically active only when exists as a homodimer, and the negative regulation of its activity by the regulatory protein antizyme (Az) is achieved via disrupting the ODC homodimer. Az can decrease polyamine levels through binding and converting ODC into an enzymatically inactive Az-ODC heterodimer. In addition, the Az-ODC heterodimer can be recognized by the 26S proteasome, which targets ODC for an ubiquitin-independent degradation. Besides interacting with ODC, Az also suppresses polyamine uptake from the extracellular environment through inhibiting polyamine transporters. Thus, an increase in Az lowers the levels of ODC and polyamine, in turn reducing cell growth. On the other hand, the expression of a catalytically dead ODC homolog named antizyme inhibitor (AzIN) increases polyamine levels by competing with ODC for Az to prevent Az-mediated ODC degradation. Together ODC, Az, and AzIN form a delicate regulatory circuit to coordinate intracellular polyamine homeostasis.Though the overall three-dimensional structures of Az-AzIN and Az-ODC complexes are similar, Az-AzIN is more stable than Az-ODC and only the latter can be recognized by the 26S proteasome. It has been suggested that small molecule- mediated interruption of Az-AzIN heterodimerization without affecting the stability of Az-ODC heterodimer may be exploited in anticancer therapy by blocking polyamine production. But our understanding of the structural differences between Az-AzIN and Az-ODC complexes have remained incomplete due to the lack of detailed structural information about the Az-AzIN complexes. Thus, the goal of my thesis research is to determine a crystal structure of Az-AzIN at high resolution.
Since previously obtained crystals formed by human AzIN (hAzIN) and an N- terminal truncated form of human Az (hAz95-228) diffract only to low resolution, we tested whether crystals of the cross-species complexes formed by mouse Az (mAz) with either mouse AzIN (mAzIN) or hhAzIN, and hAz with mAzIN may produce better diffraction data. We have successfully established a protein expression system for producing large amount of mAzIN-hAz95-228 complex and used immobilized metal affinity, ion exchange and gel filtration chromatography for purification. Using vapor diffusion crystallization technique, various conditions for growing mAzIN- hAz95-228 crystals have been identified. However, only low resolution diffraction data were obtained from these crystals. Different post-crystallization treatments, including crystal dehydration and cryo-protection procedures, will be employed to improve the diffraction quality of these crystals.

並列關鍵字

polyamine ； ornithine decarboxylase ； antizyme ； antizyme inhibitor

參考文獻

1. Bachrach, U., The early history of polyamine research. Plant Physiology and Biochemistry, 2010. 48(7): p. 490-495.

2. Miller-Fleming, L., et al., Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell. J Mol Biol, 2015. 427(21): p. 3389-406.

3. Park, M.H., E.C. Wolff, and J.E. Folk, Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. Biofactors, 1993. 4(2): p. 95-104.

4. Xie, X., M.E. Tome, and E.W. Gerner, Loss of Intracellular Putrescine Pool- Size Regulation Induces Apoptosis. Experimental Cell Research, 1997. 230(2): p. 386-392.

5. Igarashi, K. and K. Kashiwagi, Characteristics of cellular polyamine transport in prokaryotes and eukaryotes. Plant Physiology and Biochemistry, 2010. 48(7): p. 506-512.