SUMO (small ubiquitin-like modifier) 蛋白質(也稱為Smt3
(suppressor of Mif2 protein 3)) ，它和ubiquitin 作用方式類似，都需要
E1 和E2 酵素的幫忙的後接上它們的目標蛋白（target proteins），使目
標蛋白被修飾。在脊椎動物中有三種SUMO 蛋白：SUMO-1、SUMO-2
和SUMO-3，而無脊椎動物中只有一種SUMO 蛋白。SUMO 和目標蛋
白接上之後就會影響目標蛋白的功能（如在細胞中的位置）、影響一些
基因的表現，或使細胞進入細胞凋亡(apoptosis)。
為了得知SUMO 在細胞中所扮演的角色，我們實驗室在之前以酵
母菌雜交實驗(yeast two hybrid assay) 中發現，SUMO-1/2/3 都會和
Daxx (Fas death domain-associated protein) 的N 端(Daxx1, 1-282 amino
acids) 和C 端(Daxx4, 607-740 amino acids) 有交互作用。為了得知
SUMO-1/2 作用(sumoylation) 在Daxx 的哪一個胺基酸上，我們做出
六種不同的突變：K60R (在Daxx1 上)、K630A、K631A、K634A、
K630,631A 和K630,631,634A (在Daxx4 上)，來找出Daxx 的sumoylation
site。
我們試管中sumoylation 實驗的結果顯示Daxx1 的K60 雖然是其中
一個sumoylation site，但是其並不是一個主要和SUMO 結合的地方，
Daxx 和SUMO 結合的地方主要是在Daxx 的C 端(Daxx4)。SUMO-1
和SUMO-2 在K630、K631、K634 的結果並不相同；SUMO-1 對於不
同的Daxx4 突變蛋白的結合能力沒有非常明顯的差異，但SUMO-2 對
於不同的突變卻有明顯的差別。所以我們得到的結論是K631 和K634
是SUMO-1 主要的sumoylation 位置，而K630 和K631 是SUMO-2 主
要的sumoylation 位置。除此之外我們也建議在Daxx 635-740 胺基酸
片段區域可能還有其他的sumoylation sites，除非SUMOs 和Daxx
635-740 胺基酸片段區域之間的sumoylation 為Daxx4 於試管中無法正
確摺疊而產生的假陽性反應(pseudo-positive results)。

SUMO (small ubiquitin-like modifier) protein, also known as Smt3
(suppressor of Mif2 protein 3) of Saccharomyces cerevisiae is an
ubiquitin-like protein due to the similar post-transcriptional modifications
to their substrates. There are three members of SUMO genes (SUMO-1,
SUMO-2 & SUMO-3) in the vertebrate, while only one SUMO gene exists
in the invertebrate. Covalent modification of cellular proteins by the
SUMO regulates various cellular processions, such as nuclear transport,
transcription repression and cellular apoptosis. To investigate the biological
functions of SUMO-1 and SUMO-2, yeast two hybrid assays were applied.
Results showed that N-terminus (Daxx1, 1-282 amino acids) and
C-terminus (Daxx4, 607-740 amino acids) of Daxx were the SUMOs
interacting fragments.
For identification of the sumoylation site on Daxx1 and Daxx4, six
mutants (K60R, K630A, K631A, K634A, K630, 631A and K630, 631,
634A) were constructed. In vitro sumoylation were applied in the Daxx1
fragment and mutated Daxx1 (K60R) as well as the Daxx4 fragment and
mutated Daxx4 (K630A, K631A , K634A, K630, 631A and K630, 631,
634A) to identify the sumoylation sites of Daxx.
Our results showed that Daxx1 K60 was one of the sumoylation sites,
neverthless it was not a major sumoylation site. The major sumoylation
sites were on the C-terminus of Daxx (Daxx4). The major sumoylation sites
of SUMO-1 on Daxx4 seemed different from those of SUMO-2. Mutants
(K631A and K634A) of Daxx4 decreased the yields of sumoylation
complexes of SUMO-1 more than that of Daxx4 K630. However, mutants
(K630A and K631A) of Daxx4 decreased the yields of sumoylation
complexes of SUMO-2 more than that of Daxx4 K634. Thus we propose
that the major sumoylation sites of SUMO-1 on Daxx are K631A and
K634A and that of SUMO-2 are K630A and K631A. Daxx may have other
sumoylation sites on the Daxx C-terminal 635-740 amino acids fragment,
unless the sumoylation reactions of Daxx mutants were pseudo-positive
reactions which might be caused by the improper folding of Daxx4 during
in vitro sumoylation.

中文摘要...............................................I
英文摘要...............................................III
壹、序論...............................................1
1. SUMO ...............................................1
1.1 SUMO-1 ............................................1
1.1.1 SUMO-1 的基因構造(gene structure) ...............2
1.1.2 SUMO-1 的蛋白質構造(protein structure) ..........4
1.1.3 SUMO-1 在細胞內所扮演的功能......................5
(1) SUMO-1 對細胞週期(cell cycle) 及DNA 修補的作用.....5
(2) SUMO-1 對RanGAP1 的作用............................6
(3) SUMO-1 對IκBα 的作用...............................6
(4) SUMO-1 對p53 的作用................................7
(5) SUMO-1 對DNA topoisomerase II 的作用...............8
(6) SUMO-1 對Mdm2 的作用...............................8
(7) SUMO-1 對PML 的作用................................9
1.1.4 SUMO-1 和疾病的關係..............................9
1.2 SUMO-2 和SUMO-3 ...................................10
1.2.1 SUMO-2 和SUMO-3 的基因與蛋白質...................10
1.2.2 SUMO-2 和SUMO-3 在細胞內的功能...................11
(1) SUMO-2、SUMO-3 可對C/EBPβ - 1 進行修飾
(sumoylation) .........................................11
(2) SUMO-3 會影響APP protein 的代謝途徑................11
(3) 在細胞遭受壓力下細胞中SUMOs 複合物的改變的情形.....12
2. Daxx (Fas death domain-associated protein) .........12
2.1 Daxx 的基因與蛋白質................................13
2.2 Daxx 在細胞中的功能................................13
2.2.1 Daxx 對於apoptosis 的影響........................13
2.2.2 Daxx 對於發育的影響..............................14
2.2.3 Daxx 和PML、HDAC的相互作用.......................14
2.2.4 Daxx 和HDAC 抑制劑的反應.........................15
2.2.5 Daxx 和染色體中央節蛋白-C 的相互作用.............15
2.2.6 Daxx 是一種轉錄抑制因子(transcriptional repressor) 15
2.2.7 MSP58 調控Daxx 的transcriptional repression ......16
2.2.8 Daxx 會被HIPK1 磷酸化.............................16
2.2.9 Daxx 和SUMO-1 的作用..............................17
3. SUMO 的修飾過程(sumoylation) ........................17
3.1 SUMO 對目標蛋白的修飾工作(sumoylation) .............18
3.2 目標蛋白上sumoylation 的位置........................19
3.3 SUMOs 的自我修飾(self-sumoylation) .................20
4 . 以酵母菌二次雜交系統探討SUMOs 與Daxx
的關係..................................................21
貳、研究目的............................................23
參、實驗方法和材料......................................24
1. 多株抗體(polyclonal antibodies)的製備................24
1.1 實驗動物、材料與藥品................................24
1.2 多株抗體製備的方法..................................25
1.3 多株抗體濃度和專一性的測試(ELISA) ..................25
1.3.1 ELISA 的藥品......................................25
1.3.2 ELISA 的方法......................................26
1.4 多株抗體專一性之測試(Western Blot) .................27
2. E.coli expression plasmids 及mutants 的製備..........28
2.1 pGEX-KG-p53 和pGEX-KG-PML primers 的設計............28
2.2 pGEX-KG-Daxx1 mutant primers 的設計.................29
2.3 pGEX-KG-Daxx4 mutants primers 的設計................30
2.4 pGEX-KG-Daxx1-ST primers 的設計.....................32
2.5 pGEX-KG -Daxx1-ST* mutant primers 的設計............33
2.6 pET- 2 8 a - SUMO- 1 -GG、pET-28a-SUMO-2-GG、
pET-28a-SUMO-1- GG △ 和pET-28a-SUMO-2- GG primers △
的設計..................................................33
2.7 pET-28a-SUMO-1* K16A 和pET-28a-SUMO-2* K11A
mutants primers 的設計..................................34
2.8 PCR overlap extension mutagenesis ..................36
2.9 PCR 反應的使用試劑量、產物長度和PCR conditions .....38
2.9.1 PCR 反應..........................................38
2.9.2 PCR 產物的長度....................................38
2.9.3 PCR condition ....................................39
2.10 DNA 電泳分析.......................................39
3. DNA cloning .........................................40
3.1 Vectors 及PCR 產物的限制酵素切段及純化..............40
3.1.1 材料及藥品........................................40
3.1.2 Vectors 及inserted DNA 所用的限制酵素.............40
3.1.3 vectors 的restriction enzyme reaction ............40
3.1.4 Inserted DNA的restriction enzyme reaction ........41
3.2 Vectors 與inserted DNA 的ligation ..................41
3.2.1 ligation 的藥品...................................41
3.2.2 Vectors 和inserted DNA 的ligation ................41
3.3 E. coli transformation .............................42
3.3.1 preparation of competent cells ...................42
3.3.2 Cloning vectors 的transformation .................43
3.4 Recombinant vectors 的初步篩選......................43
3.4.1 實驗材料及儀器....................................43
3.4.2 實驗方法..........................................43
3.5 Recombinant vectors 的確認(DNA sequencing) .........44
3.5.1 Preparation of autosequence gel ..................44
3.5.2 DNA sequencing analysis ..........................45
4. Protein expression, purification and storage ........47
4.1 Protein expression .................................47
4.1.1 實驗材料..........................................47
4.1.2 實驗方法..........................................48
4.1.3 IPTG induction times .............................48
4.2 Protein purification ...............................49
4.2.1 純化蛋白質的材料..................................49
4.2.2 純化蛋白質的方法..................................49
5. Sumoylation assay ...................................50
5.1 in vitro sumoylation assay .........................50
5.1.1 實驗材料..........................................50
5.1.2 實驗方法..........................................51
5.2 SDS-PAGE ...........................................51
5.2.1 實驗材料..........................................51
5.2.2 SDS-PAGE 膠片之製備...............................52
5.2.3 Sumoylation products 的電泳方法...................53
6. Western blot ........................................53
6.1 實驗材料............................................53
6.2 實驗方法............................................54
肆、結果................................................55
一. SUMOs 的自我修飾反應(self-sumoylation) .............55
二. SUMOs 對Daxx1 的修飾反應(sumoylation assays) 的
探討....................................................59
2.1 SUMOs 對Daxx1 的修飾反應............................59
2.2 SUMOs 對Daxx1* (K60A 突變蛋白) 的修飾反應...........62
2.3 SUMO-1 對Daxx1 ST (較短片段) 的修飾反應.............65
2.4 SUMOs* 對Daxx1 ST* (K60R 突變蛋白) 的修飾反應.......68
2.5 SUMOs*- Daxx1 ST* 修飾反應產物的定量................71
三. SUMOs 對Daxx4 的修飾反應(sumoylation assays)的探
討......................................................76
3.1 SUMOs 對Daxx4 的修飾反應............................76
3.2 SUMOs 對Daxx4* (K630-634A 單點突變蛋白)
的修飾反應..............................................79
3.3 SUMOs 對Daxx4* (K630-634A 多重突變蛋白)
的修飾反應..............................................81
3.4 SUMOs*-Daxx4*突變蛋白修飾反應產物的定量.............86
伍、討論................................................94
1. SUMO-1 和SUMO-2 self sumoylation ....................94
2. Identification of the sumoylation site in Daxx1 .....96
3. Identification of the sumoylation site in Daxx4 .....97
4. SUMO 對於Daxx 的影響.................................100
5. 未來的研究方向.......................................100
陸、參考文獻............................................101
柒、附錄................................................111
(一) 突變DNA 的定序結果.................................111
(二) pGEX-KG 載體的map .................................123
(三) pET-28a 載體的map .................................124
(四) 蛋白質的胺基酸序列.................................125
(五) 多株抗體(Polyclonal antibody) ELISA 反應及western blot
測試....................................................127
(六) 蛋白質的純化.......................................142
表目錄
Table 1：不同物種間SUMO-1/2/3 蛋白質的相似度和相同度....3
Table 2：Expression vectors 的fusion proteins 之長度....46
Table 3：Expression vectors 的fusion proteins 之預估的分子量......................................................47
Table 4：Daxx1 ST、Daxx1 ST*、p53 和SUMO-1* 形成sumoylated
products 的定量.........................................72
Table 5：Daxx1 ST、Daxx1 ST*、p53 和SUMO-2* 形成sumoylated
products 的定量.........................................74
Table 6 (A)：Daxx4 wild type、Daxx4 mutants 和SUMO-1* 形成
sumoylated products 的定量..............................88
Table 6 (B)：Daxx4 wild type、Daxx4 mutants 和SUMO-1* 形成
sumoylated products 的定量..............................89
Table 7 (A)：Daxx4 wild type、Daxx4 mutants 和SUMO-2* 形成
sumoylated products 的定量..............................91
Table 7 (B)：Daxx4 wild type、Daxx4 mutants 和SUMO-2* 形成
sumoylated products 的定量..............................92
圖目錄
Fig 1. 不同物種的SUMO 胺基酸序列相關性..................3
Fig 2. SUMO-1 的結構圖..................................4
Fig 3. Conjugation pathway of ubiquitin and SUMO .......18
Fig 4. PCR overlap extension mutagenesis ...............37
Fig 5 (A)：Western blot of self sumoylation assay ......57
Fig 5 (B)：Western blot of self sumoylation assay ......58
Fig 6 (A)：Western blot of Daxx1、p53 和PML sumoylation assay ..................................................60
Fig 6 (B)：Western blot of Daxx1、p53 和PML sumoylation assay ..................................................61
Fig 7 (A)：Western blot of Daxx1、Daxx1*K60R 和p53 sumoylation assay.......................................63
Fig 7 (B)：Western blot of Daxx1、Daxx1*K60R 和p53 sumoylation assay.......................................64
Fig 8 (A)：Western blot of Daxx1 ST、p53 和PML sumoylation assay ..................................................66
Fig 8 (B)：Western blot of Daxx1 ST、p53 和PML sumoylation assay ..................................................67
Fig 9 (A)：Western blot of Daxx1 ST、Daxx1 ST* K60R 和p53 sumoylation assay.......................................69
Fig 9 (B)：Western blot of Daxx1 ST、Daxx1 ST* K60R 和p53 sumoylation assay ......................................70
Fig 10：Daxx1 ST、Daxx1 ST* K60R、p53 和SUMO-1* K16A sumoylation assay 產物的定量結果........................73
Fig 11：Daxx1 ST、Daxx1 ST* K60R、p53 和SUMO-2* K11A sumoylation assay 產物的定量結果........................75
Fig 12 (A)：Western blot of Daxx4 sumoylation assay ....77
Fig 12 (B)：Western blot of Daxx4 sumoylation assay ....78
Fig 13：Mono-sumoylation assay of Daxx4 mutant proteins 80
Fig 14 (A)：Mono-sumoylation assay of Daxx4 wild type and Daxx4 mutants ..........................................82
Fig 14 (B)：Mono-sumoylation assay of Daxx4 wild type and Daxx4 mutants ..........................................83
Fig 15 (A)：Mono-sumoylation assay of Daxx4 wild type and Daxx4 mutants ..........................................84
Fig 15 (B)：Mono-sumoylation assay of Daxx4 wild type and Daxx4 mutants ..........................................85
Fig 16：Daxx4 wild type、Daxx4 mutants 和SUMO-1* K16A sumoylation assay 產物的定量結果........................90
Fig 17：Daxx4 wild type、Daxx4 mutants 和SUMO-2* K11A sumoylation assay 產物的定量結果........................93

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