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研究生:陳次郎
研究生(外文):Tzu-Lang Chen
論文名稱:探討GNMT與NPC2蛋白之交互作用
論文名稱(外文):A Study on the Interaction between GNMT and NPC2 proteins
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:公共衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
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:77
中文關鍵詞:甘胺酸氮甲基轉移酶尼曼匹克症C型二號蛋白脂肪堆積膽固醇運輸脂肪代謝異常黃體激素
外文關鍵詞:glycine N-methyltransferaseGNMTNiemann-Pick disease Type C2NPC2cholesterol traffickinghalf-life of NPC2progesterone
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甘胺酸氮甲基轉移酶 (glycine N-methyltransferase, GNMT)是大量表現在肝臟的肝癌腫瘤抑制基因。其功能主要是調控S-adenosylmethione與S-adenosylhomocysteine的比例,並與葉酸結合,進而影響甲基化與DNA的穩定,GNMT也會與環境致癌物結合並參予細胞解毒。為更了解GNMT在細胞內的其他功能,利用酵母菌雙雜交系統發現GNMT會與尼曼匹克症C型二號蛋白(Niemann-Pick disease Type C2, NPC2)結合。NPC2蛋白會與未酯化膽固醇結合,並參與膽固醇代謝。當NPC2蛋白基因變異,導致膽固醇代謝異常,累積在溶小體中,臨床上會造成肝脾腫大、神經退化與脂肪堆積。GNMT基因剔除鼠會自發性產生肝癌和脂肪代謝異常的疾病。因此,本研究目的在於證實GNMT與NPC2的交互作用,並找出交互作用主要的結合區,進一步探討GNMT與NPC2結合對細胞內膽固醇運輸的影響。利用免疫沉澱法證實GNMT與NPC2做結合,而由間接免疫螢光染色顯示,GNMT主要表現在細胞質內,在溶小體內觀察不到GNMT螢光影像;而NPC2蛋白主要表現在溶小體裡,但細胞質也可以觀察些許的表現並與GNMT螢光影像重疊。另外溶小體萃取分離實驗也證實NPC2蛋白會表現在細胞質與溶小體中。由此結果顯示GNMT與NPC2的交互作用位置應在細胞質中。更進一步探討GNMT與NPC2在細胞內參與膽固醇代謝的運輸機制。當細胞在無脂肪的狀況下,NPC2雖然分佈在溶小體裡但訊號減弱,若添加低密度脂蛋白後,NPC2在細胞內訊號增強,並且有從溶小體往外擴散的情形。當黃體激素存在下會使NPC2會大量堆積在溶小體內,而去除黃體激素後,NPC2又從溶小體往外擴散。當細胞大量表現GNMT,給予低密度脂蛋白與黃體激素處理,發現對NPC2的分布情形並無差異。利用蛋白質合成抑制劑處理與脈衝追蹤技術發現,GNMT的存在會使NPC2蛋白的半衰期顯助延長。除此之外,利用即時定量聚合酶連鎖反應去分析NPC2在人類肝癌組織的表現情形,結果顯示NPC2在肝癌組織與非肝腫瘤組織的表現並沒有差異。本研究證實GNMT主要是藉由其C端第171-295的胺基酸位置與NPC2的第81-105胺基酸位置做結合,GNMT使NPC2蛋白的半衰期顯助延長在細胞內穩定度,以維持細胞內膽固醇的代謝與運輸。
Glycine N-methyltransferase (GNMT) is a tumor suppressor gene for liver cancer. It affects DNA methylation and DNA stability by regulating the ratio of S-adenosylmethionine to S-adenosylhomocysteine and binding to folate. GNMT also serves as a 4S PAH-binding protein and participates in detoxification pathway. Recently, Niemann-Pick C2 protein (NPC2) was identified as a GNMT interaction protein using yeast two-hybrid system. Niemann-Pick C disease is an inherited autosomal recessive disorder. Deficiency NPC2 gene in NPC disease results in accumulation of unesterified cholesterol in the lysosome. NPC2 is a small soluble 151 aa glycoprotein found in the lumen of lysosomes and plays an important role in cholesterol homeostasis. In this study, we will characterize the interaction between GNMT and NPC2 and identify the interacted domains. In addition, we will clarify the hypothesis that GNMT interacts with NPC2 to maintain cholesterol trafficking and homeostasis inside the cells.
Co-immunoprecipitation assay demonstrated that the C-terminal (171-295 a.a) of GNMT interacted with C conserved region (81-105 a.a) of NPC2. Indirect-immunofluorescene (IFA) and lysosome isolation analyses showed that GNMT was expressed in the cytosol but not in the lysosome. NPC2, on the other hand, was mainly expressed in the lysosome. It is worthy to note that NPC2 also can be detected in the cytosol.
To study the role of GNMT and NPC2 in cholesterol trafficking, IFA combined with confocal microscopy assays were used to detect the GNMT and NPC2 protein localization following low density lipoprotein (LDL) and progesterone treatment. Although NPC2 was mainly expressed in lysosome under lipoprotein-deficient condition, the re-distribution of NPC2 from lysosome to cytosol was observed after addition of LDL treatment for 24 h. Progesterone, which blocks cholesterol transport out of lysosome, keep NPC2 in the cholesterol-laden lysosome. Following removal of progesterone from the media for 6-12 h, the NPC2 staining of lysosomes was lost. Instead the NPC2 was trafficking into the cytosol. After progesterone was washed out for 18-24 h, NPC2 became enriched in the lysosome. Over-expressed GNMT did not alter the NPC2 distribution following LDL and progesterone treatment.
Protein synthesis inhibitor (cycloheximide) treatment showed that the half-life of NPC2 was 1.4 h, whereas over-expressed GNMT enhanced the NPC2 protein stability to 3.5 h. Pulse-chase experiments also confirmed that GNMT could increase NPC2 protein stability form 2.6 h to 4.6 h. We also treated the WT and Gnmt-/- mice with cycloheximide and found that Gnmt-/- mice had higher NPC2 degradation rate than the WT mice.
Although GNMT is down-regulated in human liver tumor (HCC) tissues, the real-time PCR analysis indicated that the expression of NPC2 was no difference between HCC and HCC-adjacent tissues. Taken together, this study demonstrates that GNMT uses its C-terminal domain (171-295 a.a) to interact with C conserved region (81-105 a.a) of NPC2. In addition, GNMT may enhance NPC2 protein stability and maintain the cholesterol trafficking inside the cells.
中文摘要 5
Absract 7
第一章 研究背景 9
甘胺酸氮甲基轉移酵素(Glycine N-Methyltransferase, GNMT) 9
尼曼匹克症(Niemann-Pick disease) 13
尼曼匹克C型二號蛋白(Niemann-Pick disease Type 2) 14
第二章 研究目的及特定目標 17
第三章 材料與方法 18
第四章 結果 30
第五章 討論 37
第六章 參考文獻 42
圖表 46
圖一、GNMT的代謝途徑 47
圖二、GNMT的拓撲學圖示 48
圖三、NPC2的結構圖示 49
圖四、pCDNA3/HA-NPC2-HA質體的構築與蛋白質表現 50
圖五、GNMT與NPC2蛋白的交互作用 51
圖六、NPC2與GNMT蛋白的交互作用 52
圖七、GNMT不同胺基酸片段與NPC2之間的交互作用 53
圖八、帶有NPC2不同片段的pNPC2-DsRed質體的構築與蛋白質表現 55
圖九、NPC2不同胺基酸片段與GNMT之間的交互作用 56
圖十、NPC2與GNMT在細胞內分佈 57
圖十一、免疫螢光染色觀察NPC2、GNMT在細胞內包器分佈情況 58
圖十二、細胞質與溶小體萃取分離 59
圖十三、NPC2蛋白在LDL與progesterone處理後的細胞內分佈情形 60
圖十四、NPC2與GNMT蛋白經過LDL與progesterone處理後在細胞內分佈 63
圖十五、NPC2 mRNA與蛋白質在野生型以及GNMT基因剔除鼠的表現量 64
圖十六、在蛋白質合成抑制劑處理下細胞內的NPC2、GNMT半衰期 65
圖十七、NPC2、GNMT蛋白在細胞內的半衰期 66
圖十八、在野生型及Gnmt基因剔除小鼠動物體內的NPC2、GNMT半衰期 67
圖十九、人類肝癌組織NPC2 mRNA的表現量 68
圖二十、NPC2 mRNA再有無肝炎病毒感染、不同肝癌病程、與肝腫瘤大小的肝癌患者組織的表現量。 69
圖二十一、NPC2與GNMT在酵母菌雙雜交系統實驗結合區域 71
圖二十二、GNMT蛋白質四級結構示意圖 72
圖二十三、NPC2蛋白質四級結構示意圖 73
圖二十四、NPC2 Ubiquitination位置預測 74
表一、NPC2序列的引子 75
表二、NPC2即時定量聚合酶連鎖反應的引子 76
表三、台灣肝癌網124肝癌病人人口學變項 77
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