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研究生:林惠儀
研究生(外文):Huei-Yi Lin
論文名稱:尋找與SOCS6交互作用之蛋白質
論文名稱(外文):Isolation of suppressor of cytokine signaling 6 (SOCS6) interacting protein
指導教授:張久瑗
指導教授(外文):Jeou-Yuan Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:79
中文關鍵詞:SOCS6交互作用酵母菌雙雜交實驗
外文關鍵詞:SOCS6interacting proteinyeast two-hybrid
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SOCS,為suppressor of cytokine signaingl的簡稱,全名為細胞激素訊息抑制因子,具有負回饋 (negative feedback) 調控細胞激素或生長因子所引發的訊息傳導功能。因與調控JAK/STAT pathway 相關,又稱為cytokine-inducible SH2 protein (CIS) 或 STAT-induced STAT inhibitor (SSI)。根據實驗室的初步研究結果,SOCS6的表現在胃癌中是明顯降低的,過度表現SOCS6會抑制細胞生長並且引發細胞凋亡(apoptosis),因此認為SOCS6是扮演抑癌蛋白質(tumor suppressor)的角色,此外過度表現SOCS6亦會造成細胞粒線體斷裂及粒線體膜電位降低的情形。根據文獻指出,SOCS6會與elongin B、elongin C 以及cullin-5結合形成ECS E3 ligase複合體,促使與SOCS6結合的蛋白質降解。為了進一步探討SOCS6的功能,在此實驗中主要是利用酵母菌雙雜交系統(yeast two-hybrid),找出與SOCS6具交互作用的蛋白質。藉由酵母菌雙雜交系統所挑出的菌落,進行序列定序、酵母菌生長分析(Growth assay)、β-galactosidase活性分析、α-galactosidase分析、3-AT (3-amino-1,2,4-triazole)篩選以及連續稀釋等方式,總共篩選出51個候選基因。接著以分子生物學方式進一步確定這些候選蛋白中OS-9、ATPIF1以及NDUFS3與SOCS6之交互作用,以in vitro GST 融合蛋白沉澱法 (pull down)以及in vivo共同免疫沉澱法(Co-immunoprecipitation)證實其與SOCS6之結合;並同時以酵母菌雙雜交技術來探討其與SOCS6結合的區域,目前初步確認SOCS6可與ATPIF1及NDUFS3結合。之後的實驗將進一步了解此交互作用如何影響 SOCS6 的功能,進而探討SOCS6調控細胞凋亡,引發粒線體形態改變之機制。
Suppressor of cytokine signaling (SOCS), also named cytokine-inducible SH2 protein (CIS) or STAT-induced STAT inhibitor (SSI), are feedback inhibitors that negatively regulate cytokine- and growth factor-mediated signaling. Recently, it has been shown that the SOCS protein can act as a component of E3 ligase complex to facilitate the degradation of its interacting proteins, including JAK and phosphorylated signaling intermediates. We have previously shown that the expression of SOCS6 is down-regulated in human gastric cancer, and overexpression of SOCS6 inhibits cell proliferation and colony formation, suggesting that SOCS6 may act as a tumor suppressor. In addition, our data also showed that ectopically expressed SOCS6 is localized in mitochondria, leading to mitochondrial fragmentation and decrease of membrane potential. To further explore the function of SOCS6, in this study, Gal4 yeast two-hybrid system is performed to isolate the interacting proteins of SOCS6. Currently, 51 genes encoding candidates of SOCS6-interacting proteins have been identified by their abilities to promote yeast growth in conditionally drop-out media. They include OS-9, ATPIF1, NDIFS3 and etc. To further confirm the interaction of SOCS6 and the target proteins, in vitro GST pull-down assays and in vivo co-immunoprecipitation assays have been performed. The interactions of ATPIF1 and NDUFS3 to SOCS6 were confirmed. The domains of SOCS6 that are involved in the interactions are determined. Futher characterization of the interaction of SOCS6 and its interacting proteins shall provide information on how SOCS6 functionally regulates cellular processes, including cell proliferation, apoptosis and mitochondrial homeostasis.
論文電子檔著作授權書 I
論文審訂同意書 II
誌謝 III
縮寫表 IV
中文摘要 VI
英文摘要 VII
目錄 VIII
第一章 緒論 1
第一節 癌症之生成 1
一、 抑癌基因( tumor suppressor ) 1
二、 致癌基因 (oncogenes) 2
三、 癌細胞之特性 2
四、 癌細胞的惡性轉型 2
第二節 SOCS蛋白質簡介 4
第三節 SOCS6蛋白質的簡介 7
第四節 粒線體動態平衡與細胞凋亡 8
一、 粒線體動態平衡 8
二、 粒線體在細胞凋亡中所扮演的角色 9
第五節 候選基因介紹 10
一、 OS-9 10
二、 ATPIF1 11
三、 NDUFS3 11
第六節 前人研究及實驗目的 13
第二章 材料與方法 13
第一節 酵母菌雙雜交技術 (yeast two-hybrid system) 13
一、 簡介 13
二、 GAL4雙雜交系統 14
三、 酵母菌株 14
四、 培養液 14
五、 酵母菌連續轉型作用 (yeast sequential transformation) 15
六、 製備正反應對照組及負反應對照組 17
七、 酵母菌生長分析 (Growth assay) 及 α-galactosidase活性分析 17
八、 β-galactosidase活性分析 17
九、 製備酵母菌質體 18
第二節 大腸桿菌勝任細胞(competent cells)之製備與轉殖(transformation) 18
一、 勝任細胞(competent cells)製備 19
二、 電衝法 (Electroporation) 19
第三節 菌落聚合酶連鎖反應 (colony polymerase chain recation;colony PCR) 19
第四節 酵母菌蛋白質製備 (Preparation of protein extracts) 20
第五節 細胞培養 (cell culture) 21
一、 細胞株 21
二、 細胞培養液 21
三、 細胞之繼代培養(subculture) 21
第六節 轉染反應 (Transfection) 21
一、 轉染試劑 (Lipofectamine 2000) 21
二、 電穿孔 (Electroporation) 22
第七節 共同免疫沈澱 ( co-immunoprecipitation;co-IP ) 22
第八節 GST融合蛋白沉澱實驗 (GST pull-down assay) 23
一、 體外轉錄及轉譯反應(TNT) 23
二、 GST融合蛋白的表現及純化 24
三、 GST pull-down 24
第九節 免疫螢光染色 (Immunofluorescence stain ) 25
第十節 西方墨點轉漬分析 (Western blot) 26
一、 SDS-PAGE 26
二、 西方墨點法 (Western blot) 26
第三章 結果 27
第一節 酵母菌雙雜交技術 (yeast two-hybrid system) 27
一、 以酵母菌表現GAL4-SOCS6融合蛋白 28
二、 以酵母菌雙雜交技術篩選SOCS6 28
三、 以酵母菌雙雜交技術確認各個候選蛋白質與SOCS6的作用區域 30
第二節 GST融合蛋白沉澱實驗 (GST pull-down assay) 31
一、 進行不同片段GST-SOCS6融合蛋白純化 31
二、 以不同片段GST-SOCS6融合蛋白與表現候選基因蛋白的結合情形 32
第三節 共同免疫沉澱實驗 (co-immunoprecipitation;co-IP) 32
一、 SOCS6融合蛋白與Elongin C的結合情形 33
二、 SOCS6融合蛋白與Myc-OS-9及Myc-ATPIF1的結合情形 33
三、 SOCS6融合蛋白與Flag-NDUFS3的結合情形 34
第四節 免疫螢光染色 (Immunofluorescence stain ) 34
第四章 討論 35
第一節 探討利用酵母菌雙雜交技術篩選SOCS6的可能影響原因 35
第二節 探討GST融合蛋白沉澱實驗 (GST pull-down assay)的可能影響原因 36
第三節 探討共同免疫沉澱實驗 (co-immunoprecipitation;co-IP)的可能影響原因 38
第四節 探討共同免疫染色 (Immunofluorescence stain) 的可能影響原因 38
第五節 未來展望 39
第五章 參考文獻 40
第六章 圖 45
第七章 表 71
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