中文摘要
粒線體呼吸鏈第一酵素複合體對於能量產生是非常重要的。其中的次單元NADH
dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) 具有高度保留性並含有第一酵素複合體
最後一個鐵硫中心N2。NDUFS7 為一個由細胞核轉錄後在細胞質轉譯的蛋白並藉由粒線體標
的序列(mitochondrial targeting sequence, MTS)進入粒線體。除此之外，在靠近NDUFS7Ｃ端
的位置上帶有核導出訊號(nuclear export signal, NES)及核導入訊號(nuclear localization signal,
NLS)。類小泛素化(SUMOylation)是指目標蛋白被類小泛素(small ubiquitin-like modifier,
SUMO)進行可逆性的修飾且被認為在調控細胞內反應上扮演關鍵性的角色。在先前的研究，
我們已經探討了SUMO1 對於NDUFS7 的修飾及其重要性。在本次的研究，我們進一步探討
其他旁系(paralogue)的類小泛素蛋白SUMO3 對於NDUFS7 的修飾及功能。為了找出主要類
小泛素化的位置，我們將SUMO3 上第11 號位置的離氨酸(Lysine)突變成精氨酸(Arginine)使
其只能形成單一SUMO3 的鍵結但無法形成多個SUMO3 鍵結的長鏈，我們也將NDUFS7 上
僅有可能與SUMO3形成鍵結的七個Lysine 及經預測最有可能形成非共價SUMO交互作用區
域(SUMO-interacting motif, SIM)各別突變進行觀察。根據突變分析的結果，沒有任何一種上
述建構的突變可以完全消除NDUFS7被SUMOylatiton 的現象。因此，NDUFS7的SUMOylation
可能同時有多個Lysine 及SIM 的參與。另外，透過NDUFS7 與SUMO3 的融合蛋白
(NDUFS7_SUMO3AA fusion protein)顯示與SUMO1 修飾相反的結果: SUMO3 的修飾會促進
NDUFS7 進入粒線體內且幾乎不會出現在核內。SUMO3 修飾對於NDUFS7 的生理意義也利
用給予不同的條件例如：破壞粒線體的膜電位、誘發細胞凋亡、缺氧、氧化壓力以及飢餓狀
態進行探討。更進一步地，我們發現SUMO3 對NDUFS7 的修飾在氯化鈷(CoCl2)誘發的缺氧
狀態會上升但在二氧化二氫(H2O2)誘導的氧化壓力時會下降。更詳細有關SUMOylation 如何
影響NDUFS7 在胞內的移動及其生理意義值得更進一步的研究。

Mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for energy
production. Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is a
well-conserved subunit and contains an iron-sulfur cluster N2 (4Fe-4S tetranuclear) as the finial
electron transport center in complex I. As a nuclear-encoded protein and translated in cytosol, we
previously demonstrated NDUFS7 is imported into mitochondria by a mitochondrial targeting
sequence (MTS). In addition, we also identified that NDUFS7 possesses a nuclear export signal
(NES) and a nuclear localization signal (NLS) near the C-terminus. SUMOylation, the process of
protein conjugation with a small ubiquitin-like modifier (SUMO), is one type of reversible
modification and has been considered to play a key role in modulating many important cellular
processes. In previous studies, we have shown that NDUFS7 could be modified by SUMO1 and the
importance of this modification has been discussed. In this study, we tried to explore other
involvement of SUMO3, another SUMO paralogue, in NDUFS7 modification and function. To
map the major SUMOylation sites, we first mutated the lysine residue at position 11 on SUMO3 to
arginine, which was used to study the mono-SUMOylation and abolished the poly-SUMOylated
chain. We also constructed a series of NDUFS7 mutants with mutation(s) at seven lysine residues
or removing the predicated SUMO-interacting motif (SIM). According to the result of
mutation-scanning analyses, SUMOylation of NDUFS7 couldn’t be fully abolished in any of the
mutant construct. Therefore, multiple lysine residues and SIMs might be involved in the
SUMOylation of NDUFS7. In addition, the change of NDUFS7 translocation was observed in
NDUFS7_SUMO3AA fusion protein. In contrast to SUMO1 modification, NDUFS7 conjugation
with SUMO3 facilitated the import of NDUFS7 into mitochondria and nearly not in the nucleus.
The biological meaning of NDUFS7 modification with SUMO3 was investigated by introducing
various treatments such as dissipation of mitochondrial membrane potential, etoposide-induced
III
apoptosis, hypoxia, oxidative stress and deprivation of nutrition, respectively. Among them, we
uncovered that SUMOylation of NDUFS7 with SUMO3 was up-regulated in the CoCl2-induced
hypoxia condition but down-regulated under H2O2-triggered oxidative stress. The detailed
mechanism about how SUMOylation influences NDUFS7 subcellular localization and its
contribution to physiologic consequences deserves further exploration.

中文摘要 ........................................................................................................ I
Abstract ......................................................................................................... II
Abbreviations .............................................................................................. IX
Introduction ................................................................................................... 1
1. Mitochondria .............................................................................................................. 1
1.1 Mitochondrial homeostasis .................................................................................... 2
1.2 The machineries and mechanisms of protein importing into mitochondria ........... 3
1.3 The crosstalk between mitochondria and the nucleus ............................................ 4
1.3.1 Mitochondrial proteins directing mitochondria-to-nucleus signaling to regulate nuclear
gene expression .................................................................................................. 5
1.3.2 Protein dual targeting to mitochondria and the nucleus in response to stress 6
1.4 Mitochondrial oxidative phosphorylation (OXPHOS) system .............................. 6
1.5 Respiratory supercomplex ...................................................................................... 7
1.6 Mammalian mitochondrial complex I: structure and function ............................... 8
1.6.1 Structure .................................................................................................... 8
1.6.2 Function .................................................................................................... 8
1.7 Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) .............. 9
1.7.1 The implication of NDUFS7 in diseases ................................................. 10
2. SUMOylation ............................................................................................................ 11
2.1 Small ubiquitin-like modifier (SUMO) proteins .................................................. 12
2.2 The SUMO pathway ............................................................................................ 12
2.3 The SUMO modification site(s) ........................................................................... 14
2.3.1 SUMO consensus motifs (SCMs) for site-directed conjugation ............. 14
2.3.2 SUMO-interacting motifs (SIMs) ........................................................... 15
2.4 The consequence of SUMOylation and its biologic meaning .............................. 15
2.4.1 SUMOylation and subcellular translocation ........................................... 16
2.4.2 SUMO and genotoxic, DNA damage response ...................................... 17
2.4.3 SUMO and oxidative stress ..................................................................... 18
2.4.4 SUMO and nutrient response .................................................................. 18
2.4.5 SUMO and hypoxia ................................................................................ 19
3. SUMOylation in mitochondria ............................................................................... 20
The object of this study .............................................................................. 21
V
Materials and methods ............................................................................... 22
Results .......................................................................................................... 33
1. The sequence alignments of NDUFS7 and its homologous species .......................... 33
2. The prediction of NDUFS7 modification by small ubiquitin-like modifier (SUMO)33
3. NDUFS7 is a substrate for SUMO3 modification ..................................................... 34
4. Multiple lysine residues are contributed to the SUMO3 modification of NDUFS7 .. 35
5. SUMO-interacting motifs (SIMs) are involved in the SUMO3 modification of NDUFS7 36
6. SUMO3 modification promotes the mitochondrial import of NDUFS7 ................... 37
7. Dissipation of mitochondrial membrane potential or apoptosis dose not have a significant
effect on SUMO3 modification of NDUFS7 ................................................................. 38
8. The level of SUMO3 modification of NDUFS7 is reduced under H2O2-induced oxidative
stress ............................................................................................................................... 39
9. Deprivation of serum does not have a significant effect on SUMO3 modification of NDUFS7
........................................................................................................................................ 39
10. An increase of SUMO3 modification of NDUFS7 is observed under hypoxia condition
induced by hypoxia-mimicking reagent CoCl2 .............................................................. 40
Discussion .................................................................................................... 41
1. The SUMOylation pattern of NDUFS7 ..................................................................... 42
2. The involvement of SUMOylation in subcellular location of NDUFS7 ................... 43
3. SUMOylation of NDUFS7 with SUMO3 and its physiologic consequences ............ 45
3.1 SUMOylation versus etoposide induced gentoxic stress and mitochondria-mediated
apoptosis ..................................................................................................................... 45
3.2 SUMOylation versus oxidative stress and mitochondrial unfolded protein response 46
3.3 SUMOylation versus starvation and mitochondria autophagy ............................ 48
3.4 SUMOylation versus hypoxia and mitochondrial ROS ....................................... 49
4. The correlation of SUMOylation and phosphorylation in NDUFS7 ......................... 51
5. The limitation of the study ......................................................................................... 52
6. Conclusion ................................................................................................................. 53
Tables ........................................................................................................... 55
Figures .......................................................................................................... 57
References .................................................................................................... 70
Appendixes .................................................................................................. 80

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