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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/71414

    Title: Involvement of Arc1p biotinylation and a WHEP domain on aaRS activity
    Authors: 張至堯;Chang,Chih-Yao
    Contributors: 生命科學系
    Keywords: 生物素化;轉錄後修飾;蛋白質合成;線蟲;神經退化;aminoacyl-tRNA synthetase;biotinylation;post-translational modification;protein synthesis;tRNA;Caenorhabditis elegans;CMT disease;neurologic disorder;WHEP domain
    Date: 2016-07-25
    Issue Date: 2016-10-13 13:02:20 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 之前的研究發現細胞質methionyl-tRNA synthetase (MetRS) 及 glutamyl-tRNA synthetase (GluRSc)會與Arc1p形成一個三元複合體,藉此增強MetRS與GluRS胺醯化tRNA的能力,另外也藉由複合體的形成調控MetRS與GluRSc在細胞內的分布,分離的GluRSc及MetRS會分別進入粒腺體及細胞核內作用。Arc1p的結構包含三個區段,N端主要是與MetRS及GluRSc結合,M及C端則組合成一個tRNA結合區段。最近的研究指出,Arc1p的N端區段有一個SSKD序列,其中K86會經由轉錄後修飾作用加上生物素(biotin),目前還不清楚生物素化對Arc1p的結構及功能有何影響。我們的研究結果顯示:在正常生長溫度下(30oC),Arc1p會被生物素化(15%),經修飾後的Arc1p仍可正常地與MetRS 及 GluRSc形成複合體,也可與tRNA結合;但是,在高溫(37oC)環境下,Arc1p幾乎不會被生物素化,未被生物素化的Arc1p較被生物素化的Arc1p更穩定,因此在高溫下能更有效率地提升GluRSc及MetRS的催化能力。或許Arc1p可藉由生物素化或去生物素化來調節其蛋白質結構及功能,對抗環境溫度的改變。
    某些真核生物的aminoacyl-tRNA合成酶(aaRSs)存在一段WHEP區段和該區段常與tRNA或蛋白質結合。我們研究發現線蟲細胞質和線粒體的Glycyl-tRNA synthetase(CeGlyRS)由相同的基因(CeGRS1)不同的起始碼轉譯而成。其結果是,細胞質形式蛋白質其N端具有WHEP區段,而其線粒體形式蛋白質具有mitochondrial targeting signal (MTS; aa 1~20) 及附加區段 appended domain (aa 21~64)。在實驗中得知兩種形式的CeGlyRS均能辨認及使用線蟲的細胞質tRNAsGly,線粒體形態的CeGlyRS比細胞質形態的CeGlyRS更能有效辨認及使用線蟲的線粒體tRNAGly(缺少TψC loop)。儘管WHEP域本身缺乏tRNA的結合能力,但去除此區段會降低了酵素的催化效率。最有趣的是,去除WHEP區段的CeGlyRS具有更高的熱穩定性和稍低的結構彈性。我們的研究顯示WHEP區段可能具有調節酵素的動態結構和活性的功能。
    ;Previous studies showed that cytoplasmic methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRSc) form a ternary complex with an aaRS cofactor, Arc1p, thereby enhancing their aminoacylation activities. In addition, Arc1p also regulates the subcellular distribution of these two associated enzymes. Upon dissociation from the ternary complex, GluRSc and MetRS are targeted into the mitochondria and nucleus for functioning. The structure of Arc1p can be divided into three domains, N, M, and C domains. The N domain interacts with GluRSc and MetRS, while the M plus C domains form a non-specific tRNA-binding domain. A recent report demonstrated that a SSKD motif in the N domain of Arc1p can be biotinylated through post-translational modification in vivo. However, the biological significance of this modification remained unclear. We show herein that Arc1p was biotinylated (15%) under normal growth conditions. However, biotinylation had little effect on its ability to interact with tRNA or GluRSc/MetRS. In contrast, Arc1p was almost biotin free at a high temperature. Non-biotinylated Arc1p was more heat-tolerant and more efficiently promoted the aminoacylation activity of GluRSc. Perhaps the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes.
    WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases (aaRSs) and play roles in tRNA or protein binding. We show herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. As a result, the cytoplasmic form possessed an N-terminal WHEP domain, while its mitochondrial counterpart possessed an extra N-terminal sequence (aa 1~64) consisting of a mitochondrial targeting signal (MTS; aa 1~20) and an appended domain (aa 21~64). Cross-species rescue assays showed that this dual-functional gene effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 (which encodes GlyRS) knockout strain. While both forms of CeGlyRS efficiently charged the cytoplasmic tRNAsGly of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNAGly isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA-binding activity, deletion of this domain reduced the enzyme’s catalytic efficiency. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests the WHEP domain may act in cis to regulate the enzyme’s dynamic structure and activity.
    Appears in Collections:[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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