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標題: 探討人類胜肽精胺酸去亞胺酶第四型的胺基酸對受質專一性
Study on the amino acid residues of human peptidylarginine deiminase 4 for substrate specificity
作者: 施凱文
Shih, Kai-Wen
關鍵字: peptidylarginine deiminase 4;人類胜肽精胺酸去亞胺酶第四型
出版社: 生命科學系所
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人類胜肽精胺酸去亞胺酶 (peptidylarginine deiminase,PAD) 為需要鈣離子存在下才有活性的酵素,在轉譯後修飾中將目標蛋白上的arginine轉變成citrulline,此反應稱為protein citrullination (deimination)。PAD4是唯一擁有nuclear localization signal (NLS) 的異構型,NLS可以幫助細胞將PAD4運送進細胞核。近年來許多研究指出PAD4與許多疾病有關,例如:類風濕性關節炎 (RA)、惡性腫瘤等。PAD4與受質結合狀態下的結構已被解出,但對於PAD4如何辨識受質仍然不清楚,為了探討PAD4對受質專一性的關係,利用定點突變修改PAD4與受質結合位置上的胺基酸,將目標胺基酸修改成alanine 和相似類型的胺基酸,以酵素動力學實驗分析突變後酵素對peptidylarginine的活性。再透過生物資訊的方法比對guanidino-group modifying enzymes 上的胺基酸,找出PAD4與arginine deiminase差異的胺基酸,並修改為相同胺基酸,然後進行酵素動力學實驗分析對 arginine活性。由實驗結果得知R372、V469、W347在PAD4上為不可取代的胺基酸,R374、R639對活性影響較小;而S406突變為aspartate和glutamate不能增加PAD4對arginine的活性。

Peptidylarginine deiminase (PAD) is a Ca+2-dependent enzyme that can catalyse the conversion of arginine residue to citrulline in protein in posttranslational modification
and the reaction is named citrullination or deimination . PAD4 is only one isotype with nuclear localization signal (NLS) that can help cell transfer PAD4 into nucleus .
Recently, some researches indicates a relationship between PAD4 and many disease including RA and cancer . The structure of PAD4 with substrate was resolved but it is still unknown how to recognize the substrate of PAD4. To study the substrate specificity of PAD4, we mutated the amino acid of substrate binding site to Ala or similar amino acids, then analysed the activity by kinetic assay . Determining the difference between PAD4 and guanidine-group modifying enzymes though bioinformation, we replaced residues of PAD4 with same residue of the other guanidine-group modifying enzymes by site-directed mutagenesis and analysed different substrate by enzyme kinetic assay. Results suggested R372, V469 and W347 cannot be substituted for other amino acids, R374 and R639 are less influential on activity;replacing S406 with Asp and Glu cannot increase the activity of PAD4 for Arg.
其他識別: U0005-1808201118525700
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