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研究生:呂律欣
研究生(外文):Lu-Hsin Lu
論文名稱:探討甘胺酸氮甲基轉移酶與核醣體蛋白S20之交互作用
論文名稱(外文):A study on the interaction between GNMT and Ribosomal protein S20
指導教授:陳宜民陳宜民引用關係
指導教授(外文):Yi-Ming A. Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
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:71
中文關鍵詞:甘胺酸氮甲基轉移酶核醣體蛋白S2018S rRNABud23
外文關鍵詞:GNMTRPS2018S rRNABud23
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甘胺酸氮甲基轉移酶 (Glycine N-methyltransferase,GNMT) 在肝臟中含量豐富。主要藉由調控S-adenosylmethionine (SAM) 和S-adenosylhomocysteine (SAH)的比例,去影響基因的穩定性,同時也是肝細胞主要之葉酸結合蛋白。從GNMT基因剔除鼠上發現,缺少GNMT可以自發性產生肝癌,故GNMT是一個肝癌的腫瘤抑制基因。為更加了解GNMT的致癌機轉,藉由酵母菌雙雜交系統 (yeast two hybrid system),發現GNMT的結合蛋白Ribosomal protein S20 (RPS20)。RPS20為核醣體40S次單元的成份之一,更有研究指出,RPS20在真核細胞中參與20S rRNA加工成為18S rRNA的步驟。
實驗室先前的研究中,定義出GNMT在酵母菌中的同源蛋白Bud23,有文獻報導指出,剔除BUD23基因會阻礙20S rRNA 加工成為18S rRNA進而影響40S成熟的晚期步驟,因此40S核醣體次單元 (ribosomal subunit) 降低,造成嚴重的生長遲緩。本研究目的將證實GNMT與RPS20的交互作用、探討GNMT對RPS20的功能所造成的影響及RPS20在肝癌腫瘤組織的表現。
在酵母菌系統中,由polysome profile assay實驗結果顯示,人類GNMT質體轉型的∆bud23酵母菌株可以看到40S 次單元及polysomes表現有部分回復的現象;並且由GST pull-down assay結果證實Bud23能夠與RPS20結合。在哺乳類細胞中,經由共同免疫沉澱及間接免疫螢光染色的實驗證實GNMT與RPS20在細胞質中形成同一個複合體,並且RPS20與GNMT結合的位置於GNMT胺基酸序列74~170,但兩者間的交互作用並不會影響RPS20的蛋白穩定性;由sucrose gradient的結果顯示GNMT會與RPS20共沉積 (cosedimentation) ,推測,GNMT可能透過與RPS20的交互作用而與pre-40S subunit、40S subunit甚至是整個核醣體結合。因此,GNMT在哺乳類細胞中,可能同樣參與18S rRNA的加工 (processing)。
利用Taiwan Liver Cancer Network所提供的124對肝癌患者腫瘤組織,來測定其腫瘤組織中RPS20的表現量,分析結果顯示,RPS20在腫瘤組織的表現量顯著的高於非腫瘤組織,並且達統計上差異 (p = 0.019)。利用臨床資料,分成不同族群,在男性患者的腫瘤組織RPS20表現量明顯高於非腫瘤組織 (p = 0.015)。並且感染HCV的肝癌患者的腫瘤組織的RPS20表現量顯著高於非腫瘤組織 (p = 0.016);此外,透過cell cycle progression的實驗得知,RPS20蛋白上升表現似乎對293T細胞生長週期無顯著的影響。
Glycine N-methyltransferase (GNMT) is abundant in liver. It affects genetic stability by regulating the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and serve as a major folate binding protein in hepatocytes. The GNMT knockout mouse developed HCC spontaneously. Therefore, GNMT has been proposed to be a liver cancer suppressor gene. In order to study the role of GNMT in hepatocarcinogenesis, we used yeast two hybrid system to identify a GNMT interacting protein, Ribosomal protein S20 (RPS20). RPS20 is a component of 40S ribosomal protein and involved in the late step of 18S rRNA processing.
Our previous study identified a yeast gene with significant homology to GNMT.
In addition, deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA and a late step in 40S maturation. In this study, I intended to prove the interaction of GNMT and RPS20、the effect on the function of RPS20 and the expression level of RPS20 in HCC tumor tissues.
In yeast system, the result of polysome profile assay showed that ∆bud23 mutant introduced with phGNMT partially rescued the reduced levels of the 40S ribosomal subunit and polysomes. In addition, GST pull-down was used to examine the interaction between Bud23 and RPS20. In mammalian cells, co-Immunoprecipitation and Indirect-Immunofluorescence assay were used to determine that GNMT and RPS20 were present in the same complex in cytoplasm. Furthermore, the amino acid 74-170 of GNMT were necessary to bind to RPS20. On the other hand, the interaction between GNMT and RPS20 showed no effect on the protein stability of RPS20.The result of sucrose gradient indicated that GNMT cosedimented with RPS20. The result raised the possibility that maybe GNMT could bind to pre-40S subunit,40S subunit even ribosome and involve in 18S rRNA processing in mammalian cells.
The real-time PCR was used to determine the mRNA level of RPS20 in 124 pairs of HCC specimen from Taiwan Liver Cancer Network. Overall, the expression levels of RPS20 in tumorous tissues were significantly higher than that in tumor adjacent tissues (p = 0.019). RPS20 was significantly up-regulated in male tumorous tissues in male group (p = 0.015). In addition, the expression level of RPS20 was up-regulated in HCV infected patients (p = 0.016). Moreover, the result of cell cycle progression assay revealed that up-regulation in RPS20 had no effect on the cell cycle progression of 293T cells.
目錄
中文摘要 7
英文摘要 9
第一章 研究背景 11
1、 甘胺酸氮甲基轉移酵素 (Glycine N-Methyltransferase, GNMT) 11
1-1.GNMT的功能 11
1-2.GNMT如何受調控 11
1-3.GNMT入核現象 12
1-4.GNMT的表現具有組織特異性 13
1-5.GNMT基因剔除鼠 (GNMT knockout mouse) 13
1-6.GNMT 蛋白結構 13
2、 Protein involved in bud-site selection; diploid mutants display a random budding pattern instead of the wild-type bipolar pattern (BUD23) 14
3、 核醣體的裝配 (Assembly of ribosome) 15
4、 Ribosomal protein S20 , RPS20 16
第二章 研究目標 17
第三章 材料與方法 18
1、 製備RPS20 PCR產物 18
2、 質體構築 18
3、 製備大量質體DNA (QIAGEN Plasmid Maxi Kit) 19
4、 細胞培養 19
5、 細胞轉染 (cell transfection) 19
6、 蛋白質定量 20
7、 西方墨點法 (Western Blot, WB) 20
8、 GST Pull-down assay 21
9、 純化GST-Bud23重組蛋白 23
10、 免疫沉澱法 23
11、 間接免疫螢光染色(Indirect-Immunofluorescence assay, IFA) 24
12、 Polysome profile assay 25
13、 TCA沉澱蛋白質 28
14、 反轉錄作用-聚合連鎖反應(RT-PCR) 28
15、 即時反應-聚合連鎖反應 (Real-time PCR) 29
16、 細胞週期行進 (cell cycle progression) 檢測 29
第四章 結果 31
Yeast system 31
1、 GNMT在功能上部分地取代Bud23 31
2、 誘導(induction)BL21產生Bud23重組蛋白 31
3、 Bud23與RPS20的交互作用 32
Mammalian system 33
1、GNMT與RPS20在細胞質中形成同一個複合體 33
1-1共同免疫沉澱 (Co-Immunoprecipitation) 33
1-2逆向的共同免疫沉澱 (Reciprocal Co-Immunoprecipitation) 33
1-3間接免疫螢光染色法(Indirect-Immunofluorescence assay, IFA) 34
2、 GNMT不同蛋白質片段與RPS20在細胞質的交互作用 34
3、 GNMT與RPS20同沉積 (cosedimentation) 於sucrose gradient 34
4、 GNMT對於RPS20蛋白質穩定性的影響 36
5、 RPS20在肝癌組織的表現量 36
6、 GNMT及RPS20對細胞週期的影響 37
第五章 討論 39
1、 探討酵母菌Bud23與人類GNMT之異同 39
2、 探討Bud23、GNMT入核與pre-40S次單元的運送 41
3、 探討Bud23、GNMT甲基化活性對18S rRNA加工的重要性 41
4、 探討GNMT與RPS20之交互作用情形 42
5、 探討RPS20對細胞週期的影響 42
第六章 結論及未來研究方向 44
第七章 參考文獻 45


圖表目錄
圖 一、 GNMT的代謝途徑及調控。 49
圖 二、 GNMT的拓樸學圖示及立體結構圖。 50
圖 三、 核醣體的裝配、40S及60S次單元的成熟及運送。 51
圖 四、 分別轉型人類GNMT及酵母菌BUD23的∆bud23酵母菌株之核醣體次單元及polysomes回復的情況。 53
圖 五、 IPTG誘導大腸桿菌BL21產生GST-Bud23重組蛋白。 54
圖 六、 GST pull-down assay證實Bud23與RPS20之間的交互作用。 ……………………………………………………………….55
圖 七 pCDNA3-HA-RPS20質體之構築。 56
圖 八、 利用共同免疫沉澱法證實GNMT與RPS20的交互作用。 …………………………………………………….…………57
圖 九、 逆向的共同免疫沉澱證實GNMT與RPS20的交互作用。 ……………………………………………………………….58
圖 十、 GNMT及RPS20在細胞內表現的位置。 59
圖 十一、 GNMT GNMT不同蛋白質片段與RPS20在細胞質的交互作用。 …………………………………………………………….61
圖 十二、 GNMT與RPS20共沉積於sucrose gradient。 62
圖 十三、 以西方墨點法分析處理cycloheximide後,GNMT及RPS20的半衰期。 64
圖 十四、 利用real-time PCR的方法偵測124位HCC病人的腫瘤組織及非腫瘤組織mRNA的表現量。 65
圖 十五、 利用real-time PCR的方法偵測肝炎病毒感染、未受肝炎病毒感染及是否吸菸的族群之HCC病人的腫瘤組織及非腫瘤組織mRNA的表現量。 66
圖 十六、 利用real-time PCR的方法偵測不同病程之HCC病人的腫瘤組織及非腫瘤組織mRNA的表現量。 67
圖 十七、 利用real-time PCR的方法偵測是否肝硬化及腫瘤大小超過、小於3公分之HCC病人的腫瘤組織及非腫瘤組織mRNA的表現量。 68
圖 十八、 利用流式細胞儀分析GNMT及RPS20對細胞週期的影響之初步結果。 70
圖 十九、GNMT參與40S次單元成熟之假設模式。………………71
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