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研究生： 李兆昌
Lee, Chao-Chang
論文名稱： 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)
指導教授： 高茂傑
Kao, Mou Chieh
口試委員: 林玉俊
Lin, Yu-Chun
張壯榮
Chang, Chuang-Rung
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 60
中文關鍵詞： 粒線體 、人類粒線體第一蛋白質複合體
外文關鍵詞： NDUFV2, TAT
相關次數： 點閱：2  下載：0 
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    • 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。

    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.

    摘要 I Abstract II Abbreviations ……………………………………………………………....IV Introduction 1 1.Mitochondria and oxidative phosphorylation (OXPHOS) 1 2.Mechanisms of protein import to mitochondria 2 3.Mitochondrial complex I 3 4.NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUFV2) subunits 4 5.Therapies for mitochondrial diseases 5 6.Cell penetrating peptides (CPPs) and human immuno- deficiency virus (HIV)-transactivator of transcription (TAT) 7 7.Mechanisms of cell entry of CPPs (including TAT) 8 8..Main purpose and value of this study 10 Materials and Methods 13 1. TAT-NDUFV2 and NDUFV2-TAT plasmid 13 2. Overexpression and purification of TAT-NDUFV2 and NDUFV2-TAT proteins 13 3. Cell culture 14 4. Transient transfection 14 5. Subcellular fractionation 15 6. Trypsin treatment 16 7. Carbonyl cyanid p-(trifluoromethoxy) phenylhydrazone treatment 16 8. Cell viability assay 16 9. High resolution clear native gel electrophoresis (HrCNE) 16 10. Complex I in gel activity assay 17 11. Immunofluorescence staining 18 12. Dynamic Complex I activity assay 18 13. Oxygen consumption assay 19 14. Western blotting analysis 20 15. Mitochondrial membrane potential analysis 20 16. Density gradient centrifugation 20 17. Statistical analysis 21 Results 23 1. Evolution and conservation of iron-sulfur cluster binding domain N1a 23 2. Mitochondrial targeting of TAT fusion proteins by transient transfection 23 3. Overexpression and purification of exogenous TAT fusion proteins from E. coli 24 4. Exogenously produced TAT fusion proteins can be deliveried into cells and targeted to mitochondria by protein transduction 24 5. Transduction of exogenously produced TAT-NDUFV2 and NDUFV2-TAT across mitochondrial inner membrane could be independent of mitochondrial membrane potential 26 6. Immunofluorescence analysis of mitochondrial delivery of TAT-containing NDUFV2 by protein transduction 27 7. A high dose of exogenous TAT fusion protein is harmful to cell viability 27 8. The NDUFV2 knockdown cell line is established by RNA interference technique 28 9. Exogenously produced TAT-containing NDUFV2 can be incorporated into mitochondrial complex I and restore the complex I activity 28 10. Exogenously produced TAT fusion proteins can recover the spectrophotometric enzyme activity of complex I of NDUFV2 knockdown cells……………………………………………………………………………...30 11. Exogenously produced TAT fusion proteins can recover the oxygen consumption rate and mitochondrial membrane potential of NDUFV2 knockdown cells………………………………………………………………...31 Discussion …………………………………………………………………. 33 Figures 37 Figure 1. Multiple sequence alignment of NDUFV2 protein with its homologues from different species. 37 Figure 2. Immunoblotting analysis of protein localization in cells transiently transfected with TAT-NDUFV2 or NDUFV2-TAT construct 38 Figure 3. Overexpression and purification of TAT-containing NDUFV2 fusion protein from E. coli 39 Figure 4. Immunoblotting analysis of protein localization of mitochondrial delivery in cells co-cultured with TAT-NDUFV2 or NDUFV2-TAT 40 Figure 5. Immunoblotting analysis of mitochondrial delivery of TAT containing NDUFV2 after treating cells with FCCP 41 Figure 6. Immunofluorescence analysis of cells co-cultured with TAT-NDUFV2 or NDUFV2-TAT. 42 Figure 7 Viability of cells co-cultured with various concentrations of TAT-NDUFV2 or NDUFV2-TAT. 43 Figure 8. Validation of the knockdown efficiency in NDUFV2 suppression cell line IF4 44 Figure 9. Mitochondrial complex I assembly and in gel activity analysis of NDUFV2 suppression cells treated with TAT-containing NDUFV2 fusion protein…….... 45 Figure 10. The effect of TAT-NDUFV2 and NDUFV2-TAT co-cultured with NDUFV2 suppression cells on complex I enzymatic activities. 47 Figure 11. The effect of TAT-NDUFV2 and NDUFV2-TAT co-cultured with NDUFV2 suppression cells on oxygen consumption rate 49 Fig 12. The effect of TAT-NDUFV2 and NDUFV2-TAT co-cultured with NDUFV2 suppression cells on mitochondrial membrane potential by TMRM……………………………………………………………………..........50 Tables ……………………………………………………………………….51 References…………………………………………...……………… ……...52 Appendixes …………………………………………………………………56

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