穿膜胜肽TAT輔助蛋白質轉導系統之開發及其運用於彌補人類粒線體第一蛋白質複合體NDUFV2次單元缺陷之功效評估

Developing a TAT –mediated protein transduction system to rescue mitochondrial complex I deficiency caused by the defect of NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2)

李兆昌(Chao-Chang Lee)

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(2018年)

摘要

NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) 是人類粒線體第一蛋白複合體(complex I)的一個次單元。它的基因坐落於細胞核的染色體上，而其蛋白質含有一個鐵硫中心N1a。N1a在complex I 中可能作為電子暫時的儲存場所，以防止過多的電子逸散至有氧的環境中而形成活性氧分子。NDUFV2目前已被證實與多種疾病有很大的關聯性，包括阿茲海默症及帕金森氏症。從HIV病毒中發現的蛋白質衍化而來的細胞穿膜胜肽transactivator of transcription (TAT)，已經被成功的用來攜帶外來的融合蛋白穿過細胞的細胞膜，並且能保持原有蛋白的功能。在這篇研究中，我們試圖利用TAT輔助蛋白質轉導系統來開發出一種治療因NDUFV2缺陷所導致Complex I功能不足的方法。這方法是將兩個融合蛋白TAT-NDUFV2和NDUFV2-TAT分別在體外經由大腸桿菌大量生產，而後再利用TAT轉導系統將其帶入細胞之中，為多做比較，產生同樣融合蛋白的載體也利用轉染的方式使其直接在細胞內表現。結果顯示無論是外來的TAT-NDUFV2或NDUFV2-TAT，都能順利的進入細胞的粒線體當中，並能正確地與其他complex I次單元組合，這顯示TAT-NDUFV2 和NDUFV2-TAT可能具有治療NDUFV2相關疾病的效果。有趣的是，此兩種融合蛋白轉導後進入粒線體是不需要粒線體膜電位的參與。為了評估其運用於治療上的可行性，我們使用NDUFV2基因表現減量的細胞株(IF4)來進行實驗。結果顯示TAT-NDUFV2 與NDUFV2-TAT 不只能顯著的增進complex I的組裝，在complex I酵素功能的分析中，也顯示具有回復的效果，其整體細胞的氧氣消耗速率及粒線體的膜電位也有極大的提升。我們的發現證實了利用TAT輔助蛋白轉導系統來開發治療complex I功能缺失或其他粒線體相關的疾病之方法是極具潛力的。

關鍵字

粒線體 ； 人類粒線體第一蛋白質複合體

並列摘要

NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is a nuclear-encoded subunit of human mitochondrial complex I. It contains a binuclear iron sulfur cluster N1a, and may play a role in temporary storage of excess electrons to prevent free radical production. Defects of NDUFV2 have been associated with Alzheimer's disease and Parkinson's disease. Cell-penetrating peptide derived from HIV-1’s transactivator of transcription (TAT) has been successfully applied as a carrier to bring fusion proteins into cells by crossing plasma membranes without compromising the biological function of proteins. In this study, we tried to develop a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFV2 defects. Two fusion proteins (TAT-NDUFV2 and NDUFV2-TAT) were exogenously expressed and purified from E. coli for transduction of human cells. In addition, similar constructs were also generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFV2 and NDUFV2-TAT could be delivered into mitochondria and correctly assembled in complex I. Interestingly, the mitochondrial import of TAT-containing NDUFV2 was independent of mitochondrial membrane potential. To explore the therapeutic application of the developed system, a NDUFV2 knockdown cell line (IF4) generated in previous studies was applied for rescuing studies. Treating with TAT-NDUFV2 not only significantly improve the assembly of complex I in IF4 cells, but also partially rescue complex I functions both in the in-gel activity assay and the complex I enzymatic activity assay. In addition, the oxygen consumption rate and mitochondrial membrane potential of IF4 cells were also greatly increased. Similar results were also observed while IF4 cells were treated with NDUFV2-TAT. Our current findings suggest a great potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency and other mitochondrial disease.

並列關鍵字

Mitochondrial complex I ； NDUFV2 ； TAT

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