- 學位論文
探討人類第四型胜肽精胺酸去亞胺酶對轉酮酶瓜胺酸化的位置與功能
Investigation of Citrullination Site and Function of Transketolase by Human Peptidylarginine Deiminase 4
摘要
人類胜肽精胺酸去亞胺酶 (human peptidylarginine deiminase, PAD) 是一種催化精胺酸轉譯後修飾為瓜胺酸的酵素，這個轉譯後修飾的反應被稱為瓜胺酸化，而瓜胺酸化與多種自體免疫性疾病有關。人類胜肽精胺酸去亞胺酶共有五種異構酶，其中第四型胜肽精胺酸去亞胺酶 (PAD4) 會隨著發炎反應的出現而增加，經由嗜中性白血球釋放到關節中，導致促發炎細胞因子的產生和免疫細胞聚集，形成免疫複合物而造成類風溼性關節炎。 轉酮酶 (transketolase, TKT) 在大多數腫瘤組織中都有表達，能使細胞能夠滿足各種不同條件下的合成代謝需求，本文想藉由瓜胺酸化的層面來觀察這兩種蛋白的關聯性。在本篇論文中首先利用PDB-PISA預測transketolase位於表面的精胺酸位點，最後挑選出六個位點進行點突變為離胺酸，並以這些突變蛋白當作受質進行酵素動力學分析偵測PAD4的活性。之後再透過LC-MS/MS分析後，鑑定出TKT共有九個瓜胺酸化位點，與預測的位點只有兩個重複。因此再針對其餘七個針對其中七個精胺酸位點進行點突變為離胺酸並同樣當作受質進行分析PAD4的活性。 根據實驗結果發現TKT可作為PAD4的受質，且相較於野生型的TKT，以突變的TKT當作PAD4的受質，PAD4的活性較低。透過酵素動力學實驗以及LC-MS/MS加以了解兩者蛋白之間的關聯性。
並列摘要
Human peptidylarginine deiminase (PAD) is enzyme that catalyzes the post-translational modification (PTM) of arginine to citrulline. The PTM reaction is called citrullination, and it is associated with various autoimmune diseases. There are five isoforms of human PADs, of which the human peptidylarginine deiminase 4 (PAD4) will increase with the occurrence of inflammation and will be released into the joints through neutrophils, resulting in the production of pro-inflammatory cytokines and the accumulation of additional immune cells, forming immune complexes that cause rheumatoid arthritis (RA). Transketolase (TKT) is expressed in most tumor tissues, which enables cells to meet the anabolic needs of various conditions. In this study, we want to discuss the relationship between PAD4 and TKT. First, we use PDB-PISA to predict the surface arginine residues of TKT, and six arginine residues were selected to perform mutagenesis, converted the arginine residue into lysine residue. Then, use these TKT mutants as substrates of PAD4 to perform enzyme kinetic assay. Besides, we also performed LC-MS/MS to identify the citrullination sites of TKT, and nine arginine residues were identified; compared to the prediction of the PDB-PISA, only two arginine residues were overlapping. Thus, the other seven arginine residues that identified by LC-MS/MS were also mutated into lysine, and as the substrates of PAD4 to perform enzyme kinetic assay to detect the activity of PAD4. According to the experimental results, TKT can be substrate of PAD4. When used transketolase mutants as substrates, the activities of PAD4 were significantly decreased. Through enzyme kinetic experiments and LC-MS/MS, we can more understand the relationship between PAD4 and TKT.