Identification of citrullination sites of antizyme by peptidylarginine deiminase 4

Abstract

人類胜肽精胺酸去亞胺酶 (human peptidylarginine deiminase，PAD) 是一種將蛋白質轉譯後修飾的酵素，能夠將目標蛋白上的精胺酸 (arginine) 轉變成瓜胺酸 (citrulline)，而此反應稱之為protein citrullination (deimination)。胜肽精胺酸去亞胺酶總共有五種異構型 (isoforms)，而第四型胜肽精胺酸以同源雙套體形式存在人體內，需要鈣離子才會有酵素活性。近年來第四型胜肽精胺酸去亞胺酶也被認為是研究類風濕性關節炎的重要蛋白，根據研究此蛋白會催化多種受質蛋白，引發人體相關免疫疾病；而近期也陸續發現第四型胜肽精胺酸去亞胺酶會在多種癌症和惡性腫瘤組織裡有大量表現，與細胞凋亡和細胞周期的停止有密切關係；為了探討第四型胜肽精胺酸去亞胺酶與疾病相關蛋白間的作用，純化與抗酶 (Antizyme，AZ) 疾病相關的蛋白，利用酵素動力學的方式測其第四型胜肽精胺酸去亞胺酶酵素活性，結果顯示與抗酶作用後具有活性並且得知R42、R81、R86、R100、R188及R199受到脫胺作用，故推測抗酶可能是第四型胜肽精胺酸去亞胺酶之受質；而抗酶在細胞內主要透過結合人類鳥胺酸脫羧酶 (Ornithine decarboxylase, hODC) 來調控多胺之濃度，因此探究其抗酶受到第四型胜肽精胺酸去亞胺酶脫胺後之功能，由培養脫胺後與多點突變型之結果皆能發現抗酶會降低抑制人類鳥胺酸脫羧酶的能力，得知抗酶受到第四型胜肽精胺酸去亞胺酶脫胺後會改變其原本生理功能。經由探討兩者間交互作用的影響，了解第四型胜肽精胺酸去亞胺酶在抗酶調控ODC表現中所扮演的角色。The peptidylarginine deminase (PAD) family is a kind of post-translational modifying enzyme that can catalyes protein citrullination (deimination) which is the conversion of protein-bound arginine to citrulline (Arg -> Cit). PAD4, one of the PAD isoform family, which is a dimeric enzyme catalyes protein in the presence of calcium. PAD4 is considered as the important enzyme for rheumatoid arthritis in the past few years. According to the research, PAD4 will initiate immunity of this disease. PAD4 recently has been found to overexpress in many cancers and malignant tissues that closely related to the apoptosis and cell cycle arrest. In order to explore the PAD4 between the role of the enzyme and disease-related role, we have purified a variety of disease-related protein to measure PAD4 enzyme activity by enzyme kinetics assay. The results showed that PAD4 can use antizyme (AZ) as the protein substrate and the kinetic data showed that R42, R81, R86, R100, R188 and R199 are critical residues to be citrullinated in AZ. For this reason, we suggested that AZ may be as a substrate for PAD4. However, AZ mainly combinated with human ornithine decarboxylase (hODC) to regulate the concentrations of polyamine in cells. Our data showed that incubation of AZ with PAD4 and multisite mutations will reduce the ability to inhibit ODC activity. Therefore, we could know that PAD4 may change AZ founction after AZ citrullination. By according to these results, we propose that PAD4 may play an important role in the regulation of AZ of ODC activity.目次 中文摘要 I 英文摘要 II 目次 III 縮寫表 IV 表目次 V 圖目次 VI 一、前言 1 二、實驗方法 5 三、結果 13 四、討論 19 五、結論 22 參考文獻 23 表 28 圖 34 附 錄 52 實驗材料 53 主要儀器與器材 55 相關藥品配置 5