PGC-1α 與 Stra13 間之交互作用

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姓名

張心瑜(Hsin-yu Chang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

PGC-1α 與 Stra13 間之交互作用
(The Interaction between PGC-1α and Stra13)

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摘要(中)

PPAR-γ coactivator-1 (PGC-1α) 是DNA轉錄因子的共同調控因子，參與在能量代謝的調控機制中，並且與肌肉纖維型態轉換有關。當細胞受到外在環境的刺激，例如:冷，PGC-1α會被大量誘導表現。在之前已被證實PGC-1α可以和PPARγ及Mef2c共同調控其下游及自身基因的活化。 Stra13在轉錄層次上大部分扮演抑制者的功能，它是一個basic helix loop helix (bHLH)的轉錄因子。這一類的轉錄因子利用鹼性的區段與DNA的固有序列結合，Stra13屬於bHLH轉錄因子的E group，但是結合的DNA固有序列卻是B group 結合的位置。在本實驗室的研究中發現，Stra13可以抑制MyoD對於PGC-1α的transactivation的能力。在我們的研究當中，想要去了解PGC-1α和Stra13兩個蛋白質之間的交互作用，以及兩者交互作用對於PGC-1α和Stra13下游調控的基因的影響。在cell free的實驗方面，已知道PGC-1α和Stra13會有很強的結合能力，我們利用GST pull down assay的方式去觀察到PGC-1α主要利用1-128 a.a.的位置和Stra13交互作用，而Stra13則是利用1-135 a.a.的位置與PGC-1α交互作用。接著在in cell的實驗方面，將針對PGC-1α下游的調控基因，藉由nuclear respiratory factor和nuclear recptor活化後，觀察PGC-1α和Stra13交互作用的影響。利用同樣的方式觀察Stra13下游基因會不會受到影響。在未來希望能建立PGC-1α-flag穩定表現之細胞株，然後以co-immunoprecipitation的方式，觀察PGC-1α與Stra13在in cell的交互作用情形。

摘要(英)

PPAR-γ coactivator-1 (PGC-1α) serve as a coregulators of DNA-binding transcription factors controlling energy metabolism. It is implicated in the muscle fiber type- switching, response to cold exposure and other cellular energy metabolisms. Interestingly, it has also been shown that PGC-1α can coactivate PPARγ- and Mef2c-dependent activation of its own gene. Stra13 is a basic helix-loop-helix (bHLH) containing transcriptional repressor of the E group but binds to the consensus motif as B group factors. In our recent study, we found that Stra13 can repress MyoD transactivation of PGC-1α promoter activity. In this study, we are interested in the interaction between PGC-1α and Stra13 on PGC-1α- and Stra13-dependent genes. In cell-free system, pull down assay indicated that PGC-1α binds to Stra13 strongly. We also defined the regions of PGC-1α (1-128) amino acid play an important role in the interaction with Stra13. And Stra13 (1-135) amino acid is the major region which interacts with PGC-1α. To go a step further, our study is to find out whether the interaction between PGC-1α and Stra13 affects the activation of PGC-1α-dependent genes by nuclear respiratory factor and nuclear receptors. Similarly, it is also of interest to know the response of Stra13 target genes to the presence of PGC-1α. In the future, we will use co-immunoprecipitation assay as an in-cell system to reveal that their interaction.

關鍵字(中)

★ PGC-1α
★ Stra13

關鍵字(英)

★ PGC-1α
★ Stra13

論文目次

中文摘要 I
ABSTRACT II
誌謝 III
縮寫與全名對照表 VIII
一、緒論 1
Ⅰ 肌肉的起源與生成 1
Ⅱ The PGC-1α family 2
a PGC-1α 2
b PGC-1β 4
c PRC 4
Ⅲ Stra13 5
Ⅳ 研究動機與目的 6
二、材料與方法 8
Ⅰ 實驗材料 8
1 細胞株 8
1-1 細胞培養 8
2 菌株 8
2-1 菌株培養 8
2-2 菌株的保存 8
Ⅱ 質體構築 8
1 pM-PGC-1α表現質體 (expression plasmid) 建構 8
1-1 插入DNA (PGC-1α)的製備 9
1-11 聚合鏈鎖反應Polymerase Chain Reaction (PCR) 9
1-12 聚合鏈鎖反應產物的修飾 9
1-13 插入 (Insert) DNA的純化 9
1-2 載體DNA的製備 10
1-21 載體DNA限制的剪切 11
1-22 載體 DNA 的 5’端去磷酸根反應 11
1-23 載體DNA的純化 11
1-24 接合反應 ( Ligation ) 11
1-25 篩選 (Screening) 11
2 pcDNA3-PGC-1α (1-186)質體的建構 12
2-1 純化步驟 12
3 pcDNA3-PGC-1α (1-292) 質體的建構 13
4 pcDNA3-PGC-1α (1-403) 質體的建構 13
5 pcDNA3-PGC-1α (1-66) 質體的建構 13
6 pcDNA3-PGC-1α (64-128) 質體的建構 13
7 pcDNA3-PGC-1α (128-196) 質體的建構 13
8 pcDNA3-PGC-1α (1-128) 質體的建構 13
9 pcDNA3-PGC-1α (64-196) 質體的建構 14
10 pcDNA3-PGC-1α (196-799) 質體的建構 14
11 pcDNA3-Stra13 (1-135)質體的建構 14
12 pcDNA3-Stra13 (1-194)質體的建構 14
13 pcDNA3-Stra13 (1-288)質體的建構 14
14 pcDNA3-Stra13 (1-51)質體的建構 14
15 pcDNA3-Stra13 (1-108)質體的建構 14
14 pcDNA3-Stra13 (52-108)質體的建構 14
16 pcDNA3-Stra13 (108-135)質體的建構 14
Ⅲ RT-PCR 15
1 Total RNA 的抽取 15
2 反轉錄反應 (Reverse Transcription) 15
3 聚合鏈鎖反應 ( Polymerase Chain Reaction, PCR ) 16
Ⅳ Real Time PCR 定量實驗 16
Ⅴ 轉染實驗 17
1 哺乳細胞雙雜交轉染實驗 17
1-1細胞培養 17
1-2轉染作用 (Transfaction) 17
1-3螢火蟲冷光活性方法 (Luciferase Activity Assay) 17
2 過渡性轉染實驗 17
Ⅵ 蛋白質標定 18
Ⅶ 蛋白質純化 18
Ⅷ GST pull down assay 19
三、實驗結果 21
1 利用mammalian two-hybrid assay觀察PGC-1α與Stra13兩個蛋白質在in cell system 中交互作用的情形 21
2 PGC-1α 主要以 (1-186 aa) 與MBP-Stra13 binding 22
3 PGC-1α (1-128) 與MBP-Stra13結合的能力最強 23
4 Stra13不同長度之deletion對GST-PGC1α結合能力 24
5 比較C3H10T1/2與C2C12兩株細胞中PGC1α表現量的差異 25
7 Overexpression PGC1α及Stra13時，對Vascular Cell Adhesion Molecule -1 (VCAM-1) promoter的影響 27
8 pPYCAGIP-PGC-1α-flag質體的建構 27
四、實驗討論 29
五、圖表 33
圖一、 pM-Stra13與pcDNA3-PGC-1α對TK-MH-100x4-LUC活性的影響 34
圖二、 PGC-1α CDS利用酵素分成不同片段示意圖 35
圖三、觀察PGC-1α不同C端deletion clones與Stra13在in vitro的結合情形 36
圖四、 PGC-1α (1-186)之間deletion clones與PGC-1α C端196-799 示意圖 37
圖五、觀察PGC-1α(1-186)之間的deletion clones與Stra13在in vitro的結合力 38
圖六、 PGC-1α (1-186)之間deletion clones與PGC-1α C端 (196-799) 示意圖 39
圖七、觀察PGC-1α (1-186)之間的deletion clones與Stra13在in vitro的結合力 40
圖八、純化GST-PGC-1α-beads 41
圖九、 Stra13 C端deletion clones示意圖 42
圖十、 Stra13 C端不同deletion clones與GST、GST-PGC-1α結合情形 43
圖十一、 Stra13 (1-135)更小片段的deletion clones示意圖 44
圖十二、 Stra13 (1-135)之間不同deletion clone對GST、GST-PGC-1α結合情形 45
圖十三、純化 MBP-Stra13 (108-135)、MBP-Stra13 (52-135) 46
圖十四、 PGC-1α及Stra13對IAP promoter活性影響及比較C2C12、 48
C3H10T1/2兩細胞株中，PGC-1α表現量 48
圖十五、 PGC-1α及Stra13對promoter活性之影響 50
圖十六、過量表達PGC-1α及Stra13時對VCAM-1 promoter轉錄活性影響 51
圖十七、 py-PGC1α-flag質體建構 52
六、參考文獻 53
七、附錄一 55
<圖一> 55
<圖二> 56
<圖三> 57
<表一> 58
<表二> 58
<表三> 59
八、附錄二 60
60
九、附錄三 61
I 溶液及試劑配方 61
II 藥品試劑 63
III 酵素和限制 63

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2010-7-28

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