探討粒線體Phenylalanyl-tRNA synthetase突變與活性的關聯性

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姓名

花初雨(Chu-Yu Hua) 查詢紙本館藏

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生命科學系

論文名稱

探討粒線體Phenylalanyl-tRNA synthetase突變與活性的關聯性
(Characterization of mitochondrial phenylalanyl-tRNA synthetase mutants related to activity)

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摘要(中)

Aminoacyl-tRNA synthetases (aaRSs)是轉譯過程必要的酵素。在真核生物細胞質phenylalanyl-tRNA synthetase (PheRS) 的結構為異型四聚體 (heterotetramer)，由兩個α subunit及兩個β subunit組成。α subunit負責胺醯化作用，催化Phe-tRNAPhe的形成，反β subunit負責編輯作用，將misactivated aa-tRNAPhe水解成胺基酸及tRNAPhe。真核生物粒線體PheRS的結構為α一元體 (monomer)。過去的研究顯示，真核生物粒線體PheRS缺少編輯作用的功能。在病人研究報告顯示，致死性胎兒腦病變與粒線體的氧化磷酸化之缺陷有關，且與人類粒線體PheRS突變有關。然而，這些突變引發致死性胎兒腦病變的機制仍不清楚。本結果顯示，人類粒線體PheRS (HsPheRSm) 突變P136H及P361L可減少胺醯化的活性，但是突變A170G則否。此外，先前的研究顯示，常用於治療帕金森氏症 (Parkinson′s disease) 之藥物dopa，會經過錯誤性胺醯化作用而產生dopa-tRNAPhe，導致接受藥物治療的病人體內提高了含有dopa的蛋白質含量。為了測試HsPheRSm突變對於錯誤的胺基酸之辨認，本研究使用phenylalanine類似物meta-tyrosine以及meta-fluoro-phenylalanine來當作受質。在體內實驗結果顯示，含有HsPheRSm突變的細胞生長在含有錯誤的胺基酸之培養基中，並沒有發現到生長上的差異。後續研究將繼續探討HsPheRSm突變與錯誤性胺醯化作用的關聯性。

摘要(英)

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes for protein translation. In eukaryote, cytoplasmic phenylalanine-tRNA synthetase (PheRS) is a heterotetramer composed of two alpha and two beta subunits. The alpha subunit of PheRS provides an active site for catalyzing the formation of Phe-tRNAPhe, whereas the beta subunit is responsible for editing misactivated aa-tRNAPhe. In contrast to the cytoplasmic PheRS, the mitochondrial PheRS is a monomer, which contains only the alpha subunit and lacks the editing activity. Previous studies showed that oxidative phosphorylation (OXPHOS) defect-related fatal infantile encephalopathy is associated with mutations of human mitochondrial PheRS (HsPheRSm). However, the mechanisms leading the encephalopathy are still unclear. We show herein that mutations of p.P136H and p.P361L impaired their aminoacylation activity, but mutation of p.A170G did not. In addition, previous studies have shown that dopa, which is the most commonly prescribed drug for the treatment of Parkinson’s disease, can be misacylated to dopa-tRNAPhe, which in turn leads to the increased levels of DOPA-containing proteins found in patients treated with this drug. To determine the amino acid specificities of these HsPheRSm mutants, we chose several phenylalanine analogues such as meta-tyrosine and meta-fluoro-phenylalanine as substrates. Our in vivo assays showed that cells possessing HsPheRSm mutants could grow on a selective medium containing these phenylalanine analogues without obvious growth defects. Research is underway to elucidate the relationships between HsPheRSm mutations and misacylation.

關鍵字(中)

★ tRNA合成酶
★ 粒線體疾病
★ 錯誤性胺醯化作用
★ 苯丙氨酸類似物

關鍵字(英)

★ aminoacyl-tRNA synthetase
★ mitochondrial disease
★ misaminoacylation
★ phenylalanine analogues

論文目次

中文摘要　Ⅰ

Abstract　Ⅱ

誌謝　Ⅲ

目錄　Ⅳ

圖目錄　Ⅶ

表目錄　Ⅷ

縮寫檢索表　Ⅸ

第一章　緒論　1

1.1　 Aminoacyl-tRNA synthetase (aaRS) 的簡介　1

1.1.1　AaRS 的功能　1

1.1.2　AaRS 的分類　2

1.1.3　原核生物與真核生物的aaRS　3

1.2　PheRS 的簡介　3

1.2.1　PheRS 的生化特性　3

1.2.2　真核生物之細胞質與粒線體PheRS的結構　4

1.2.3　人類細胞質 (HsPheRSc) 及粒線體PheRSs (HsPheRSm)與疾病的關聯性　4

1.3　研究目的　6

第二章　材料與方法　7

2.1　菌株、載體及培養基　7

2.1.1　菌株　7

2.1.2　載體　7

2.1.3　培養基　8

2.2　大腸桿菌勝任細胞的製備與轉型作用　10

2.2.1　大腸桿菌勝任細胞的製備　10

2.2.2　大腸桿菌勝任細胞的轉型作用 (transformation)　11

2.3　酵母菌勝任細胞的製備與轉型作用　11

2.3.1　酵母菌勝任細胞的製備　11

2.3.2　酵母菌勝任細胞的轉型作用　12

2.4　質體之選殖　12

2.5　點突變 (site-directed mutagenesis)　13

2.6　酵母菌單套MSF1剔除株 (knockout strain) 的建構　14

2.7　功能性互補分析 (complementation)－測試細胞質功能　15

2.8　功能性互補分析－測試粒線體功能　15

2.9　生長性表現型分析 (growth phenotype)－測試細胞質的生長差異　16

2.10　生長性表現型分析－測試粒線體的生長差異　16

2.11　蛋白質製備 (protein preparation)　16

2.12　Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 之

蛋白質分子量分析　18

2.13　西方點墨法 (Western blotting)　19

2.14　大腸桿菌融合蛋白質的表現和純化　20

2.15　細胞外轉錄 (in vitro transcription)　23

2.16　胺醯化作用分析 (aminoacylation)　25

第三章　結果　27

3.1　HsPheRSm突變不影響蛋白質的表現量　27

3.2　HsPheRSm突變P136H及P361L減少胺醯化作用的活性，突變A170G則

無影響　27

3.3　HsPheRSm突變P136H及P361L減弱酵母菌的生長　28

3.4　HsPheRSm突變在酵母菌的生長情形比較　28

3.4.1　錯誤的胺基酸之選擇　28

3.4.2　在酵母菌粒線體的生長情形比較　29

3.4.3　在酵母菌細胞質的生長情形比較　30

第四章　討論　31

4.1　高變異性的突變必須伴隨著高度保守性才能影響HsPheRSm的胺醯化作用　31

4.2　未完全受損的胺醯化作用仍可維持細胞生長　31

4.3　錯誤的胺基酸併入蛋白質與疾病的關聯性仍未確定　32

4.4　未來目標　33

4.4.1　探討HsPheRSm突變與疾病的關聯性　33

4.4.2　探討HsPheRSm突變為何造成錯誤性胺醯化　33

參考文獻　35

圖表　39

附錄　63

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指導教授

王健家(Chien-Chia Wang)

審核日期

2015-8-25

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