臺灣博碩士論文加值系統

人類鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 主要是負責體內多元胺的生合成第一步，此也是速率決定步驟，ODC需形成同源雙聚體 (homodimer) 才具有催化功能，而當細胞中的多元胺過度表現時，會誘發抗酶 (antizyme, AZ) 表現，AZ蛋白能和ODC會結合形成AZ-ODC異源雙聚體 (heterodimer)，將ODC帶至26S蛋白酶體 (26S proteasome) 進行不需泛素參與的降解 (ubiquitin-independent degradation)，進而抑制ODC來控制體內多元胺的濃度。同時人類抗酶抑制子 (antizyme inhibitor, AZI) 會表現並解救出ODC，因AZI對AZ的親和力強於AZ對ODC的親和力，因此能解開AZ-ODC聚合體，使ODC不被降解。在文獻上，AZI和ODC在結構上或是在胺基酸序列上都有極高的相似度，差別在於AZI沒有ODC的催化活性，在降解方面，AZI是進行泛素參與降解的路徑 (ubiquitin-dependent degradation)，跟ODC進行的AZ-dependent降解路徑不太相同，因此本篇研究主旨是在探討什麼因素造成AZI和ODC降解路徑不同。首先針對AZI結構表面上的胺基酸序列來看對AZI降解的影響，從database搜尋出AZI結構表面上幾個可能為ubiquitin結合的位點: K47、K63、K125、K140、K175、K203、K327和K341，利用alanine scanning的方式把Lysine (K) 改為Alanine (A)，進行細胞外降解分析 (in vitro degradation assay) 來看野生型AZI (AZI-WT) 以及突變型 (mutant) AZI 的降解程度是否不同。經實驗後發現，把K47、K125、K140、K175這幾個位點改掉後，AZI的降解現象就減緩了，推測這幾個位點可能為AZI的泛素化位點 (ubiquitylation sites)。之後對應到ODC結構上，發現這幾個位點都不是lysine，分別是D47、N125、M140、R175，後續我們將ODC上這幾個位點改回成lysine，探討是否ODC就是缺乏了這幾個lysine，因此才無法進行泛素參與的降解路徑。實驗結果發現，當把這四個位點改回lysine後，ODC就可以進行泛素參與的降解路徑。因此我們推測在演化上有可能是因為ODC缺乏了這幾個lysine，因此才演化出AZ-dependent降解路徑。

Ornithine decarboxylase (ODC) is an important enzyme that can catalyze the ornithine into putrescine, which is the first step of polyamines production. The elevated concentration of polyamines can induce antizyme (AZ) over-expression. Then, AZ can bind with ODC and take it to 26 S proteasome through an ubiquitin-independent degradation pathway. At the same time, there is a protein can rescue ODC, named antizyme inhibitor (AZI). AZI can bind with AZ because AZ-AZI binding affinity is better than AZ-ODC binding affinity, AZI can prevent ODC from 26S proteasome degradation by forming AZ-AZI complex. Therefore, ODC can produce polyamines continually. The elevated polyamines can lead cells to transform, even worse it will become the cancer cells, therefore, the concentration of polyamines needs to be regulated. In previous study, AZI and ODC have many similarities in their structures and sequences, but the degradation pathways of them are completely different. In contrast to ODC degradation, the degradation of AZI is ubiquitin-dependent and does not interact with AZ. In our research, first, we will focus on finding out the ubiquitination sites of AZI. We selected eight lysines on AZI surface by some database and changed these lysines into alanines to check whether it interferes AZI degradation. Finally, we found that the degradation of some AZI mutants seem to be retarded. Therefore, we suggest that K47, K125, K140, and K175 may be the possible ubiquitination sites for AZI. In contrast, these sites on ODC are D47, N125, M140, R175. So, we change these sites back to lysine to check whether ODC can go through an ubiquitin-dependent degradation pathway. After experiments, we found that ODC mutants really can be degradaed by ubiquitylation. Based on the above results, we presumed that ODC lacks these lysine residues for ubiquitylation so it evolves into AZ-dependent degradation pathway in the evolution.

摘要 i
Abstract iii
縮寫表 vi
表目錄 viii
圖目錄 ix
一、前言 1
二、實驗方法及步驟 10
I. Mutagenesis 10
II. 純化 13
III. In vitro degradation assay and immunoblotting 17
IV. ODC酵素活性與受AZ抑制能力分析 21
IV.實驗流程圖 25
三、結果 26
1.資料比對和AZI單點突變 26
2. AZI-WT及8個mutant AZI蛋白質表現與純化 26
3.NaCl濃度提高能提升AZI降解效率 27
4.AZI單點突變後減緩了AZI的降解現象 27
5.AZI多點突變後減緩了AZI的降解現象 28
6.提升rabbit reticulocyte lysate並無法加速 AZI 的降解 29
7.去除AZI的N端區域並不會影響AZI的降解現象 29
8.加入突變型Ubiquitin和MG132後明顯看到AZI ubiquitylation現象 30
9.ODC多點突變後，可以促使ODC進行ubiquitin-dependent降解 30
10.ODC多點突變後，依然能走AZ-dependent降解途徑 31
11.ODC多點突變後，ODC的活性下降且易受AZ抑制 31
12.AZI多點突變後，並無法走AZ-dependent降解途徑 32
四、討論 33
1. rabbit reticulocyte lysate的不穩定性影響到AZI降解 33
2. AZI與AZ降解速率差異 33
3. AZI與ODC降解現象的穩定性 33
4. AZI和ODC的ubiquitylation現象 34
5. AZI與AZ結合後，AZI的ubiquitylation現象不明顯 34
6. AZI與AZ結合後，AZ的降解現象不明顯 35
7. ODC和AZI第125號和第140號胺基酸決定與AZ的結合親和性 35
8.決定ODC與AZI降解途徑的關鍵 36
五、結論 38
六、參考文獻 39
七、表與圖 45
附錄 72

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