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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Title:	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
Authors:	花初雨;Hua,Chu-Yu
Contributors:	生命科學系
Keywords:	tRNA合成酶;粒線體疾病;錯誤性胺醯化作用;苯丙氨酸類似物;aminoacyl-tRNA synthetase;mitochondrial disease;misaminoacylation;phenylalanine analogues
Date:	2015-08-25
Issue Date:	2015-09-23 10:52:59 (UTC+8)
Publisher:	國立中央大學
Abstract:	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。;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.
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