Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) 是人類粒線體第一蛋白質複合體中具高度保留性的一個蛋白。研究指出，NDUFS7會鍵結於一個[4Fe-4S]的鐵硫中心（iron-sulfur cluster N2, tetranuclear），其功能為擔任第一蛋白質複合體中電子傳遞的最後電子接受者。NDUFS7的基因編碼於細胞核的基因體內，在細胞質轉譯出蛋白質後才會進入粒線體，並組裝於第一蛋白質複合體的親水區塊。多數表現於粒線體基質的蛋白質是在細胞質中合成，接著再經由mitochondrial targeting sequences (MTSs)帶領其往粒線體移動並進入粒線體內行使特定功能。
在先前的研究當中，我們定義出NDUFS7上位於N端的60個胺基酸是一段有效的MTS。我們同時也發現NDUFS7的C端含有一段可能的nuclear localization signal (NLS) 以及一段可能的 nuclear export signal (NES)。這些結果顯示NDUFS7在細胞內有著細胞核和粒線體兩種分佈。關於調控蛋白在細胞核和細胞質間移動的機制，sumoylation近年來被認為扮演著很重要的角色。Small ubiquitin-related modifiers (SUMOs)在細胞質轉譯合成後，可以在E1 (SAE1/SAE2), E2 (UBC9) 和E3酵素的協助下鍵結到目標蛋白並進一步調控其下游反應。在這篇研究中我們利用預測軟體發現NDUFS7有許多可能會和SUMO鍵結的氨基酸序列，因此，我們在HEK293細胞裡面共同大量表現NDUFS7, SUMO-1 和UBC9，並且利用西方墨點法去偵測NDUFS7和 SUMO-1在細胞內是否結合。結果顯示，就上述的實驗條件下，NDUFS7可以被SUMO-1進行sumoylation。為了進一步確認實驗結果，我們接著在細胞內再一次共同表現NDUFS7， SUMO-1，UBC9以及SUMO-specific protease (SENP)，並發現在大量表現SENP時，我們利用抗體所偵測到的NDUFS7和SUMO-1的結合大幅減少，再一次證明NDUFS7的確會經由SUMO-1調控其sumoylation。此外，在本篇研究中，我們也確認NDUFS7上第202個胺基酸-Lys202是一個主要和SUMO-1蛋白鍵結的位置並且吻合傳統的sumoylation motif，同時我們也發現這個位置(Lys 202)的sumoylation對於NDUFS7蛋白的穩定性有著重大影響。然而根據目前的實驗數據仍然無法確立NDUFS7 sumoylation的確切的目的及功能，接下來的研究必須針對這部分的課題進一步做更深入的探討。

Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of the most conserved core subunits of mitochondrial complex I. NDUFS7 has a bound iron-sulfur cluster N2 (tetranuclear) which is the terminal redox center in the electron transport chain (ETC) of complex I. NDUFS7 protein is encoded by the nuclear genome and is incorporated in the peripheral segment of complex I. Most mitochondrial matrix proteins are synthesized in the cytosol and imported into mitochondria by mitochondrial targeting sequences (MTSs).
We previously defined the N terminus of the first 60 amino acids of NDUFS7 is an effective MTS. We also identified that there is a nuclear localization signal (NLS) and a nuclear export signal (NES) located in the C-terminus of NDUFS7. Sumoylation has been recognized to play an important role in protein nucleocytoplasmic transportation. Small ubiquitin-related modifiers (SUMOs) could be conjugated to target proteins by E1 (SAE1/SAE2), E2 (UBC9) and E3 enzymes after translation. Using sequence predication software, NDUFS7 protein was found to have several potential sumoylation sites. In this study, we co-transfected plasmids expressed NDUFS7, SUMO-1 and UBC9 into HEK293 cells, and detected the conjugation of NDUFS7 with SUMO-1 by western blotting with different antibodies. The results indicated that NDUFS7 could indeed be sumoylated at the current experimental conditions in vivo. To confirm these findings, SUMO-specific protease (SENP) was co-expressed with NDUFS7, SUMO-1 and UBC9 proteins in some experiments. The results showed that overexpression of SENP could reduce the level of NDUFS7 and SUMO-1 interaction. These results suggested that NDUFS7 can be sumoylated by SUMO-1 in cells. In addition, we also identified one of the sumoylation sites of NDUFS7 to be Lys 202, which is consistent with the consensus sumoylation motif and is required for NDUFS7 protein stability. Further studies are needed and on the way to investigate the functional detail of NDUFS7 sumoylation.

摘要.................................................... I
Abstract............................................... III
Abbreviations.......................................... VIII
Introduction........................................... 1
A. Mitochondrion....................................... 1
B. Sumoylation......................................... 9
C. The effects of sumoylation on biological processes.. 13
D. Mitochondrion and sumoylation....................... 18
The objective of this study............................ 20
Materials and methods.................................. 21
Result................................................. 28
NDUFS7 is a sumoylation substrate of SUMO-1............ 28
NDUFS7 has more than one accepter site for sumoylation ? ........................................................30
Identification of Lys 202 is one of the sumoylation accepter sites.................................................. 31
Sumoylation does not alter nuclear localization of LacZ- NDUFS7199-213 constructs............................... 32
Sumoylation of NDUFS7 at Lys 202 does not alter its mitochondrial translocation............................ 33
Sumoylation increases NDUFS7 protein stability......... 35
Discussion............................................. 37
The sumoylation accepter sites and the potential SUMO-interacting motif (SIM) on NDUFS7...................... 37
The biological significance of NDUFS7 sumoylation...... 40
The NDUFS7 and SUMO-2/3................................ 42
Tables................................................. 44
Figures................................................ 46
Reference.............................................. 57
Appendixes............................................. 64

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