人類粒線體(mitochondria)第一蛋白複合體(complex I)次單元的缺陷會引發多種嚴重疾病，例如賴博氏遺傳性視覺神經症(Leber hereditary optic neuropathy)和萊氏症候群(Leigh syndrome)。由於目前的傳統療法對於大多數遺傳性粒線體疾病僅能達到緩和病情的效果，有鑑於此，發展更確實與便利的新療法是迫切需要的。
先前研究指出，將蛋白和HIV之穿膜胜肽TAT連接後之重組蛋白能夠不受細胞膜的限制進入細胞內，並保持原蛋白的生化活性。本研究旨在結合TAT與粒線體領導序列(leader sequence)之功能，期望此結合能夠成功將粒線體蛋白由細胞外運輸至粒線體內以彌補原先蛋白之缺陷。由於在人類粒線體第一蛋白複合體次單元的缺陷中，最早被發現與引發萊氏症候群有直接關係的是NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8)，因此在本篇論文中，我們以NDUFS8為範例進行研究，目標是開發用於人類粒線體第一蛋白複合體缺陷的新治療方法。
目前在本論文的研究結果中顯示，不論將TAT放在NDUFS8的N或C端(TAT-NDUFS8 or NDUFS8-TAT)都能夠成功的將蛋白從細胞外運送到粒線體中，此外這兩種蛋白也都能夠被轉化成為成熟蛋白(mature protein)。研究中也發現TAT-NDUFS8和NDUFS8-TAT進入粒線體的方式不是經由已知的粒線體外膜轉移蛋白(translocase of the outer membrane)/粒線體內膜轉移蛋白(translocase of the inner membrane)所調控的途徑。另外，為了模擬NDUFS8蛋白缺陷以及加入TAT-NDUFS8後的回復狀況，我們利用本實驗室所建構之大量抑制NDUFS8表現量的細胞株(shRNA-C3)進行功能分析實驗。結果顯示加入TAT-NDUFS8後能夠完全回復shRNA-C3細胞株之人類粒線體第一蛋白複合體中NDUFS8的蛋白組成。此外，shRNA-C3細胞株在粒線體第一蛋白複合體活性和耗氧量實驗中分別提升30%和79%的功能活性。另一方面，我們發現TAT-NDUFS8進入細胞之後，能夠促使核內體(endosome)靠近粒線體並形成endosomes-mitochondria juxtaposition的現象，我們推測此現象是TAT-NDUFS8進入粒線體的途徑，此現象發生的同時，TAT-NDUFS8能夠從核內體被傳送到粒線體之中。
總結來說，在本論文研究中，我們提出了TAT-NDUFS8從細胞外到細胞內進入粒線體的可能途徑，此外，歸因於功能分析回復的實驗結果，本論文研究也能夠作為一個成功的範例以應用於未來粒線體疾病治療方法上。

Defects in subunits of mitochondrial complex I are associated with severe diseases, including Leber hereditary optic neuropathy and Leigh syndrome. However, to date, conventional treatment for the majority of genetic-based mitochondrial diseases can only be palliative. Therefore, developing a reliable and convenient treatment approach is in an urgent need.
Fusion of the protein transduction domain (PTD) of HIV-1 transactivator of transcription (TAT) with proteins has been demonstrated to bring proteins into cells by crossing plasma membranes while retaining the biological activity of proteins. In this study, we tried to apply the protein transduction concept of TAT with the mitochondrial-targeting capability of the specific leader sequence to generate a therapeutic protein delivery system which can specifically carry target proteins into mitochondria. Here, NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8), the first complex I subunit linked to Leigh syndrome, was used as the model subunit to test our specific aims, with a hope that this newly developed method could become a novel treatment for complex I deficiency.
Currently, our findings showed that both exogenously produced TAT-NDUFS8 and NDUFS8-TAT could be delivered into mitochondria and processed into the mature forms of NDUFS8. We also showed that the mechanism of TAT-NDUFS8 and NDUFS8-TAT entering mitochondria is not through the well-recognized translocase of the outer membrane (Tom) /translocase of the inner membrane (Tim) mitochondrial import pathway. Furthermore, in order to mimic the rescue of complex I deficiency, a NDUFS8 expression knockdown cell line (shRNA-C3) was used in functional analyses as the therapeutic model. Treating with TAT-NDUFS8 could completely restore the assembly of complex I in shRNA-C3 cells, and the respiratory rate of these NDUFS8 knockdown cells was also increased about 31% and 79% in the in-gel activity assay and oxygen consumption assay, respectively. Moreover, we demonstrated that when cells were cultured with TAT-NDUFS8, endosomes were found to be retrieved in close proximity to mitochondria, indicating that TAT-NDUFS8 may enter mitochondria via the endosomes-mitochondria juxtaposition.
In conclusion, our findings provide both the possible mechanism of TAT-NDUFS8 entering mitochondria and the model for therapeutic treatment of mitochondrial disorders.

摘要 i
Abstract iii
Introduction 1
1. Basic characteristics of mitochondria 1
2. Oxidative phosphorylation system 2
3. Protein import machinery of mitochondria 2
4. Mammalian mitochondrial complex I and related diseases 3
5. Human NADH dehydrogenase (ubiquinone) Fe-S cluster protein 8 5
6. Therapeutic approaches for treatment of mitochondrial diseases 8
7. Protein therapeutics 9
8. Protein transduction domain (PTD) and human immunodeficiency virus (HIV)-transactivator of transcription (TAT) 10
9. Proposed cell entry mechanisms of TAT 11
10. Targeting protein to mitochondria using TAT 13
11. Main purpose and value of this study 16
Materials and Methods 18
1. Molecular cloning of NDUFS8 18
2. Overexpression of TAT-NDUFS8 and NDUFS8-TAT proteins 19
3. Purification of TAT-NDUFS8 and NDUFS8-TAT proteins 19
4. Cell culture 20
5. Transient transfection 20
6. Delivery of proteins into cells 21
7. Subcellular fractionation 21
8. Trypsin treatment 22
9. Carbonyl cyanid p-(trifluoromethoxy) phenylhydrazone treatment 22
10. Total cell lysate 22
11. Immunoblotting 23
12. Cell viability MTT assay 24
13. High resolution clear native gel electrophoresis (HrCNE) 24
14. Oxygen consumption measurement 25
15. Immunofluorescence staining 26
16. Density gradient centrifugation 27
17. Statistical analysis 27
Results 28
1. Evolutionary conservation of iron-sulfur cluster binding domain 28
2. The fusion protein derived from transient transfection of TAT-NDUFS8 cannot enter mitochondria correctly 28
3. The fusion protein derived from transduction of TAT-NDUFS8 can enter mitochondria 29
4. Transduction of exogenously produced TAT-NDUFS8 and NDUFS8-TAT across mitochondrial inner membrane could be independent of mitochondrial membrane potential 30
5. Overloading of exogenously produced NDUFS8-TAT is harmful to cell viability 30
6. The successful knockdown of NDUFS8 gene expression by RNA interference is demonstrated at the protein levels 31
7. Exogenously produced TAT-NDUFS8 can incorporate with mitochondrial complex I and partially restore the activity of complex I 32
8. The pathway of mitochondrial targeting of exogenously produced TAT-NDUFS8 is elusive 34
9. Exogenously produced TAT-NDUFS8 may enter mitochondria via the endosomes-mitochondria juxtaposition 35
Discussion 38
Table
Table 1. Oligonucleotide sequence of primers 52
Figures 54
Figure 1. Multiple amino acid sequence alignment of NDUFS8 with its homologues from different species 54
Figure 2. Immunoblotting analysis of protein localization and pathway of mitochondrial delivery in cells transiently transfected with various TAT-containing constructs 55
Figure 3. Immunoblotting analysis of protein localization of mitochondrial delivery in cells cultured with TAT-NDUFS8 or NDUFS8-TAT 56
Figure 4. Immunoblotting analysis of the pathway of mitochondrial delivery in cells cultured with TAT-NDUFS8 or NDUFS8-TAT 57
Figure 5. Cell viability of cells cultured with various concentrations of TAT-NDUFS8 or NDUFS8-TAT 58
Figure 6. The knockdown effect of RNA interference on protein levels of NDUFS8 59
Figure 7. Mitochondrial complex I assembly and in-gel activity assay in T-REx-293, shRNA-C3 and shRNA-C3+TAT-NDUFS8 cells by HrCNE and immunoblotting 61
Figure 8. Measurement of cellular oxygen consumption in T-REx-293, shRNA-C3 and shRNA-C3+TAT-NDUFS8 cells by Mitocell S200 micro respirometry system 62
Figure 9. Colocalization analysis of the TAT-NDUFS8 with mitochondria and endosomes 64
Figure 10. Immunoblotting analysis of endosomes-mitochondria juxtaposition in cells cultured with TAT-NDUFS8 65
Figure 11. Immunofluorescence analysis of endosomes-mitochondria juxtaposition in cells cultured with TAT-NDUFS8 67
Reference 68
Appendixes 75

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