探討biotin protein ligase的受質專一性

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姓名

陳怡安(I-An Chen) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討biotin protein ligase的受質專一性

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摘要(中)

生物素（維生素H）是由植物和大多數原核生物合成，是所有生物體所必需的，生物素通常作為參與二氧化碳轉移的酵素輔助因子。生物素蛋白連接酶（Biotin protein ligase；BPL），也稱為高羧酸酶合成酶(Holocarboxylase synthetase；HCS)或BirA，是負責將生物素共價連接到受質蛋白質上的酶，此蛋白質修飾雖然普遍存在所有生物，且是必要，卻是屬於比較罕見的轉譯後修飾，通常一個物種內只有1～5個蛋白質受質。BPL將生物素鍵結到受質蛋白質上特定的賴胺酸，這個賴胺酸通常位於一段保守序列AMKM中，因此生物素化具有高度專一性。Arc1p是一個Saccharomyces cerevisiae酵母菌非專一性tRNA結合蛋白質，在細胞質內與glutamyl-tRNA synthetase及methionyl-tRNA synthetase形成三聚體，除能增強此二酵素的胺醯化活性，也能調控他們的細胞內分布。有趣的是，Arc1p缺少生物素化必要的保守序列AMKM，但它卻可以被BPL1生物素化，且多數酵母菌種的BPL1無法生物素化其Arc1p，最近的研究報告發現，Arc1p包含一個獨特的生物素化位點SSKD。本研究具體目標是想找出酵母菌BPL及其受質的專一性部位，藉由Protein Data Bank中的晶體結構模型得知，AMKM及SSKD的結構差異頗大，進一步利用點突變實驗發現， SSKD附近的胺基酸序列對於生物素化很重要，改變這些胺基酸會明顯降低其生物素化的程度，將Arc1p的SSKD突變成AMKM後，Arc1p就不再能被生物素化。另外，透過序列比對，我們發現在S. cerevisiae酵母菌BPL1的 C端有一些獨特的嵌入胜肽，這些獨特的胜肽序列不存在多數酵母菌BPL1中，然而刪除這些獨特的胜肽序列並不會影響其生物素化Arc1p的能力。除此之外，透過不同酵母菌種之間交換BPL1片段，也無法準確定位酵素專一性區域，目前仍有待進一步的研究去解開酵母菌BPL1的專一性謎團。

摘要(英)

Biotin (vitamin H), an essential coenzyme synthesized by plants and most prokaryotes, is required by all organisms. Biotin serves as a cofactor for enzymes that participates in carbon dioxide transfer. Biotin protein ligase (BPL), also known as holocarboxylase synthetase (HCS) or BirA, is the enzyme responsible for attaching biotin to proteins. Although this modification is ubiquitous and necessary for all organisms, a relatively rare post-translational modification. Only one to five protein acceptors in every single species. BPL catalyzes the attachment of biotin to a specific lysine residue located within a conserved sequence AMKM. Arc1p is a non-specific tRNA-binding protein of Saccharomyces cerevisiae. It forms a ternary complex in the cytoplasm with glutamyl-tRNA synthetase and methionyl-tRNA synthetase, and enhances the aminoacylation activity of these two enzymes. In addition, Arc1p also regulates the intracellular distributions of these two enzymes. While Arc1p lacks an AMKM motif, it can be specifically biotinylated by BPL1. In contrast, BPL1s of most yeast species cannot biotinylate their own Arc1p homologues. Recent studies have found that Arc1p contains a unique biotinylation site, SSKD. The aim of this project is to map the substrate-specific site of yeast BPL. According to the crystal structure models in Protein Data Bank, the structures of AMKM and SSKD are quite different. Site-directed Mutagenesis further showed that amino acid sequences flanking SSKD are important for biotinylation, and altering these amino acids significantly reduces the biotinylation level. Arc1p could not be biotinylated when its SSKD was mutated to AMKM. Also, we found that there are some unique embedded peptides in the C-terminal domain of S. cerevisiae BPL1. These unique peptides are absent ftrom most yeast BPL1 homologues. However, deleting these unique peptide sequences did not affect its ability to biotinylate Arc1p. In addition, exchange of N and C domains between BPL1s of different yeast species failed to locate the substrate-specific region accurately. Further research is underway to map the substrate-specific site of yeast BPL1.

關鍵字(中)

★ 生物素蛋白連接酶
★ 生物素
★ 生物素化

關鍵字(英)

★ Arc1p
★ AMKM motif
★ SSKD motif

論文目次

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vii
縮寫檢索表 viii
第一章緒論 1
1-1生物素(Biotin)的簡介 1
1-1-1生物素化(biotinylation) 1
1-2 Holocarboxylase synthetase (HCS) 簡介 3
1-3 Biotinidase(生物素輔酶)簡介 4
1-4 Arc1p簡介 4
第二章材料與方法 6
2-1 菌株、載體及培養基 6
2-2 大腸桿菌勝任細胞的製備與轉型作用 7
2-2-1大腸桿菌勝任細胞的製備 8
2-2-2大腸桿菌轉型作用 (transformation) 8
2-3 酵母菌勝任細胞的製備與轉型作用 9
2-3-1酵母菌勝任細胞的製備 9
2-3-2酵母菌轉型作用 9
2-4 質體之選殖 10
2-5 點突變 (site-directed Mutagenesis) 10
2-6 功能性互補試驗 (complementation) 11
2-7 蛋白質純化 (protein preparation) 12
2-8 SDS-PAGE之蛋白質分子量分析 13
2-9 西方墨點法 (Western Blotting) 14
2-10 蛋白質穩定度測試（cycloheximide-chase assay） 15
第三章結果 16
3-1 人類HCS的專一性與功能 16
3-2 Arc1p序列突變對於biotinylation的活性影響 16
3-3 酵母菌BPL1的專一性與功能 17
3-4 酵母菌BPL1突變對於biotinylation活性的影響 18
3-5 Biotinylation對Arc1p穩定性的影響 20
第四章討論 21
4-1 人類HCS能取代酵母菌BPL1的功能 21
4-2 Arc1p序列突變對於biotinylation的影響 21
4-3 酵母菌BPL1突變對Arc1p biotinylation的影響 22
4-4 Biotinylation不會影響Arc1p的細胞內穩定性 23
參考文獻 24
圖表 28
Plasmid/primer list 43

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指導教授

王健家

審核日期

2018-1-12

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