Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) 為人類粒線體中第一酵素複合體44個次單元當中的其中一個次單元。NDUFS7序列1-60的胺基酸被認定為一段有效的mitochondrial targeting sequences (MTS)，我們也發現此蛋白質C端末尾序列含有一段可能的nuclear localization signal (NLS) 以及一段可能的nuclear export signal (NES)。NDUFS7作為粒線體第一酵素複合體中電子傳遞鍊的末端，有一特殊的胺基酸序列(motif): CCXXE(X)60C(X)30CP，其在各種物種上具有高度的保留性，此motif能夠與帶有N2 [4Fe-4S] cluster 的鐵硫中心相接，成為重要的氧化還原中心。在先前的研究中我們發現NDUFS7為sumoylation 的受質。Sumoylation為一種轉譯後修飾，SUMO蛋白會透過E1酵素的活化與E2 酵素 (UBC9) 相接，再透過E3酵素或者獨立將SUMO蛋白轉移到受質上。Sumoylation的相關研究指出，受sumoylation修飾的蛋白能參與相當多樣的細胞生物過程，例如:蛋白質的運輸、活性、及對不同壓力的反應等。
在此研究當中，我們讓NDUFS7蛋白、SUMO-1蛋白和E2酵素UBC9大量表現於HEK293細胞中，再透過分離出胞器的方式探討sumoylation在細胞當中修飾NDUFS7的位置與蛋白運輸之間的關聯，並且施予多種壓力誘導劑 (例如:氯化鈷、雙氧水、無血清培養液和細胞凋亡誘導試劑) 來探討其影響。從結果中可以發現，被SUMO-1修飾的NDUFS7能夠在細胞核與細胞質中被觀察到。另外，在施予氯化鈷所引起的缺氧情況下，NDUFS7的sumoylation也顯著增加。此外，我們也建構了NDUFS7-SUMO-1的質體並將其用於表現合成蛋白。其結果顯示此合成蛋白無法進入粒線體當中。基於本研究的發現，NDUFS7的sumoylation對於此蛋白質運輸以及其生理意義機制需要更深入的探討。

Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of 44 subunits in mitochondrial complex I. The N-terminal 1-60 amino acids of NDUFS7 have been defined as a mitochondrial targeting sequence (MTS) and the C-terminus contains a nuclear localization signal (NLS) and a nuclear export signal (NES). The sequence of NDUFS7 is highly conserved in the motif: CCXXE(X)60C(X)30CP. It can bind to an iron-sulfur cluster (a [4Fe-4S] cluster) called N2, a redox center in the terminus of complex I electron transfer pathway. In previous study, we identified NDUFS7 as a protein substrate of sumoylation. Posttranslational modifications of proteins by the small ubiquitin-like modifier (SUMO) have been found to be associated with various cellular processes. The SUMO protein can be conjugated to a target protein by sequential actions of enzyme E1 (SAE1/2), enzyme E2 (UBC9) and E3 ligases. The proteins conjugated by SUMO could be involved in subcellular localization, function or responding to stresses.
In this study, NDUFS7, SUMO-1 and UBC9 were co-expressed in HEK293 cells to clarify its subcellular localization by cell fractionation. The result showed that the sumoylated NDUFS7 were present in both the nuclear and cytosol fractions. Then, we established a new construct which can express the NDUFS7-SUMO-1 fusion protein, and found that the fusion protein can block the mitochondrial import. Furthermore, the effect of different stress response on sumoylation of NDUFS7 was explored by treating cells with various stress inducers, such as CoCl2, H2O2, starvation and apoptosis reagents. The results suggested that CoCl2-induced hypoxia increases the sumoylation of NDUFS7. The effects of NDUFS7 sumoylation on the subcellular localization of the protein and its physiological consequence would be further explored.

中文摘要 I
Abstract II
Abbreviations V
Introduction 1
A. Mitochondrion 1
1.Mitochondrial structure and function 1
2.Mitochondrion oxidative phosphorylation system 2
3.Mitochondrial genome and nuclear-encoded mitochondrial proteins 3
4.Mitochondrial complex I structure and electron transfer 3
5.Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 4
6.The NDUFS7 deficiency and its related diseases 5
B. Sumoylation 7
1.Small ubiquitin-like modifier (SUMO) proteins 7
2.The sumoylation cycle 8
3.The sumoylation site 9
4.The effect of sumoylation on cellular processes 10
5.Sumoylation and protein subcellular transport 11
6.The sumoylation response on cellular stress 12
7.Sumoylation and mitochondria 15
The objective of this study 16
Materials and methods 17
Results 26
1.NDUFS7 has multiple sumoylation sites and the major modified site is Lys202 residue 26
2.Sumoylated NDUFS7 is present in both the nuclear and the cytosol fractions 27
3.Ectopically expressed NDUFS7(K202R)-SUMO-1AA fusion protein is majorly located in the cytosol fraction 28
4.The treatment of hypoxia-mimicking reagent CoCl2 increases the level of NDUFS7 sumoylation 29
5.Oxidative stress does not have a significant effect on sumoylation of NDUFS7 30
6.The level of NDUFS7 sumoylation is not significantly changed under the starvation-stress condition induced by serum reduction 31
7.Apoptosis and mitochondrial membrane potential depolarization slightly affect NDUFS7 sumoylation 31
Discussion 33
Tables 38
Figures 39
Figure 1. The triple Lys mutation of NDUFS7 can not complely abolish the sumoylated form NDUFS7. 39
Figure 2. Sumoylated NDUFS7 is present in both the nuclear and the cytosol fractions. 40
Figure 3. Ectopic expression of NDUFS7(K202R)-SUMO-1AA fusion protein and its subcellular locolization. 42
Figure 4. The treatment of hypoxia-mimicking reagent CoCl2 enhanced sumoylation of NDUFS7. 44
Figure 5. H2O2-induced oxidative stress does not have a significant effect on sumoylation of NDUFS7. 46
Figure 6. The level of NDUFS7 sumoylation is not significantly changed under the starvation-stress condition induced by serum reduction. 48
Figure 7. Both etoposide and carbonyl cyanide 4-phenylhydrazone treatment slightly affect NDUFS7 sumoylation. 50
Reference 51
Appendixes 58

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