氧化磷酸化系統是真核細胞中最重要的ATP製造來源，而第一蛋白質複合體是這系統中參與電子傳遞的第一個酵素複合體。此複合體內由粒線體基因表現所產生之次單元發生變異時可能跟某些粒線體疾病的生成有關聯性。例如ND3的缺陷可能造成：Leigh氏童年期腦脊髓病變和致命性嬰兒期粒線體疾病。不幸地，現今並沒有有效的方法可以治療由粒線體基因組缺陷所衍生的疾病。最近，同素異位基因表現法（allotopic expression）似乎是一個很有希望的方法可以用來克服上述粒線體疾病治療的盲點。利用此策略，粒線體的基因必須被重新編碼使其能在細胞質中表現，而其基因產物的N端前面必須加上粒線體標的序列來幫助它們運送回粒線體。在本篇研究中，我們測試了這方法於人類第一蛋白質複合體ND3次單元的應用性。我們合成兩個基因片段COX4MTSND3和COX8MTSND3並分別將其插入於一個具有四環黴素調控機制的質體來同素異位地表現ND3。 結果顯示重新編碼的ND3基因可以透過四環黴素的誘導而在T-REx 293細胞中表現。不過我們所挑選的兩個粒線體標的序列COX4MTS跟COX8MTS卻無法將所表現的ND3運送到粒線體內正確的位置。這失敗的粒線體運送可能歸因於ND3的高疏水性進而阻礙其運送機制。相反地，這個誘導系統成功地運用於由細胞核基因表現所產生的第一蛋白複合體次單元NDUFS7（PSST）上。我們建議在重新編碼的基因上加上其他適合的粒線體標的序列和加入3’端未轉譯區域 (3’-UTR)，或許能夠增進ND3的正確運送。從這觀點上，我們推測使用同源於ND3衣藻NUO3（由細胞核基因表現所產生）的粒線體標的序列將有助於完成「ND3同素異位基因表現」此項深具挑戰性的研究。

The oxidative phosphorylation system (OXPHOS) is the most important source of ATP production in eukaryotic cells, and the complex I is the first enzyme complex in this system which participates in the process of electron transfer. Defects in the mitochondrial-encoded subunits in complex I, such as ND3, have been associated with certain kinds of mitochondrial diseases including Leigh’s syndrome (LS) and lethal infantile mitochondrial disease. Unfortunately, there is currently no effective therapeutic approach to treat diseases derived from defects in the mitochondrial genome. Recently, allotopic expression has appeared to be a promising approach to overcome the blind spot of mitochondrial disease treatment. In this strategy, the mitochondrial gene must be recoded for being expressed in cytoplasm. To facilitate the importation back to mitochondria, mitochondrial targeting sequences (MTSs) should be added in front of the N-terminal region of the gene products. In this study, we examined the applicability of applying this approach for human complex I ND3 subunit. Two gene constructs, COX4MTSND3 and COX8MTSND3, were synthesized and individually inserted into the tetracycline-regulated vector for allotopic expression of ND3 by transient transfection. We showed that the recoded ND3 gene fragment could be expressed through tetracycline induction in T-REx 293 cells. Nevertheless, the two chosen MTSs, COX4MTS and COX8MTS, were not competent in importing ND3 to the correct location in the mitochondria. The unsuccessful mitochondrial import may be attributed to the high hydrophobicity of ND3 which may form barriers for the import machinery. In contrast, the nuclear-encoded gene for NDUFS7 (PSST) subunit of complex I was successfully employed in this induction system. We suggest that by adopting other suitable MTSs and addition of a 3’ untranslated region (3’-UTR) in the recoded mitochondrial gene may improve ND3 mitochondrial import. From this point of view, using a mitochondrial targeting sequence from a nuclear-encoded Chlamydomonas reinhardtii NUO3 (a ND3 homologue) may have the potential in accomplishing this challenging work.

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