Studies on the import into mitochondria of the human ND4L subunit by reengineered nuclear genes

改造人類粒線體ND4L基因及其蛋白質產物運送之探討

莊凱惇(Kai-Tun Chaung)

指導教授 ： 高茂傑

國立清華大學/生命科學院/分子醫學研究所/碩士(2008年)

摘要

ND4L是一個由粒線體基因表現所產生的蛋白質，並且為電子傳遞鏈中位於粒線體內膜上第一蛋白質複合體成分中的一最小次單元。ND4L發生缺陷時會導致賴博氏遺傳性視覺神經症(LHON)，然而至今對此病症仍無有效的治療法。為了提供一套可以用來探討ND4L在功能上與致病生理學上所扮演角色的方法，我們採取「同素異位法」(allotopic expression)來表現經改造後的ND4L基因，並且成功地建構了P1、S9、SOD2(16A)和SOD2(16V)等四種不同的粒線體標的序列來幫助目標蛋白使其能運送入粒線體中。另外，我們也嘗試加入SOD2的3’UTR (SOD23’UTR)於上述建構基因的衍生物中來促進將ND4L的正確運送。實驗結果顯示，這些粒線體標的序列確實能幫助較親水性的EGFP蛋白質送入粒線體，但無法將我們目標蛋白ND4L正確地送入粒線體內。其次，我們也發現SOD23’UTR的加入對ND4L運送入粒線體的幫助也是非常有限。在一些轉殖的細胞中帶有P1或S9粒線體標的序列的ND4L被發現部分黏附在構成細胞骨架成分之一的vimentin上，而部分帶有SOD2粒線體標的序列的ND4L卻被送往細胞核並且塞在細胞核膜上。分析ND4L的氨基酸序列可以發現此蛋白質具有很高比例的疏水性氨基酸，而此高度疏水性的特質可能是阻礙其被送入粒線體正確位置的主要原因。最後，由於屬於單細胞綠藻類的衣藻其ND4L基因存在於細胞核中而非於粒線體內，因此物種的ND4L可能具有粒線體標的序列來達成正確的蛋白質運送。所以透過衣藻ND4L粒線體標的序列的運用及研究也許可以幫助我們達成「促進ND4L同素異位基因表現及正確粒線體運送」此項具有挑戰性的目標。

關鍵字

粒線體 ； 同素異位法 ； 粒線體標的序列 ； 疏水性 ； 運送 ； 賴博氏遺傳性視覺神經症

並列摘要

ND4L, the smallest subunit of the hydrophobic membrane segment of complex I, is encoded by the mitochondrial genome. Defects in this subunit have been associated with Leber Hereditary Optic Neuropathy (LHON) diseases. In spite of its importance, no effective remedies have been established for ND4L deficiencies. To provide a way for investigating the functional and pathophysiological role of the ND4L subunit, we adopted the allotopic expression strategy and successfully constructed and synthesized the recoded ND4L genes with the conjugation of P1, S9 and SOD2(16A) or SOD2(16V) mitochondrial targeting sequence (MTS). In addition, we also generated several derivatives with the SOD2 3’ untranslated region (SOD23’UTR) from the aforementioned constructs to facilitate mitochondrial import. The results demonstrated that these newly designed MTSs were doing their jobs in helping EGFP protein in the process of mitochondrial import. However, the precursor proteins of our targeted ND4L were not completely import competent. The addition of SOD23’UTR in the transgenes only had a very limited success in contranlational mitochondrial import. Interestingly, some TREx-293 cells expressed ND4L with P1MTS or S9MTS addition were partially associated with vimentin. In contrast, some expressed ND4L proteins with SOD2MTS were stuck on the surface of nuclei. Analyses of the contents of ND4L amino acid sequence suggested that the highly hydrophobic characteristics of this protein may hamper its correct mitochondrial import. Finally, we suggested that mitochondrial targeting sequences from Chlamydomonas reinhardtii ND4L which is nDNA-encoded may help us to achieve this challenging task.

並列關鍵字

ND4L ； allotopic expression ； mitochondrial targeting sequence ； hydrophobicity ； import ； Leber Hereditary Optic Neuropathy

參考文獻

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