張震東Chang, Geen-Dong臺灣大學:生化科學研究所陳亭靜Chen, Ting-ChingTing-ChingChen2010-05-042018-07-062010-05-042018-07-062009U0001-3107200912293300http://ntur.lib.ntu.edu.tw//handle/246246/178909三氧化二砷是一種環境中的致癌物，可能造成人體許多疾病，然而，它也是臨床上的抗癌藥，可應用在治療急性骨髓白血病；在細胞中，它會去引起細胞內的毒性，產生活性氧自由基去攻擊核酸，蛋白質，脂質，以及粒線體，進而造成細胞凋亡，同時也會產生內質網壓力以及促使壓力顆粒的形成。經由先前實驗室成員利用蛋白質體鑑定Transglutaminase II (TG 2)的受質發現，TG2的受質中含有許多已知壓力顆粒的蛋白，因此本論文擬探討，TG2是否在壓力顆粒中扮演催化壓力顆粒形成的角色。因此我們藉由三氧化二砷來研究壓力顆粒與TG2之間的關係。利用免疫螢光染色觀察到TG2的部分受質會出現在壓力顆粒的組成中，包含有APG-1，Tom34和PDIA4，這些分子都是新發現的壓力顆粒組成成分，也由此結果推測TG2在壓力顆粒上可能是扮演協助壓力顆粒組成的角色。然而，卻從in vivo和in vitro transamidation反應發現三氧化二砷會攻擊TG2酵素上的thiol group (SH)，使TG2催化活性喪失，這說明了在三氧化二砷處理下細胞內TG2催化活性受到抑制，也排除掉TG2催化壓力顆粒形成的可能性。另外，在研究中利用免疫螢光染色發現了處理三氧化二砷的Cos-1細胞中，TG2會跑到粒線體結構中，並且圍繞在細胞核和MTOC周邊，同時也會讓MFN-1和MFN-2的表現量上升，推測此現象為粒線體融合反應。由細胞流式儀發現這樣的粒線體融合反應會伴隨著活性氧自由基的生成和粒線體膜電位的下降，也會讓細胞色素C由粒線體做釋放，進而造成細胞走向細胞凋亡，這樣的現象提供了新的研究方法去研究粒線體融合反應與細胞凋亡之間的關聯性。而使用共同免疫沉澱法發現三氧化二砷的處理會使TG2與Grp78之間的結合力增加，這個現象，除了說明Grp78與TG2之間有結合作用外，還代表著在氧化壓力下，Grp78可能會成為TG2的chaperone。以上這些現象都是相當有趣的，但目前我們還無法確定TG2在粒線體融合反應上所扮演的角色。Arsenic trioxide is an environmental carcinogen which causes diseases in humans. However, it has often been used as an effective treatment for acute promyelocytic leukemia and has the potential to provide a cure for solid malignant tumors. Arsenic trioxide induces cellular toxicity by increasing the level of reactive oxygen species which target DNA, proteins, lipids and mitochondria, resulting in apoptosis. Arsenic trioxide has also been shown to induce ER stress and the formation of stress granules. Using the proteomic approach, we have previously identified the substrates of transglutaminase II (TG2) which include many known components of stress granules. To determine whether TG2 could catalyze the formation of stress granules, we used arsenic trioxide for inducing cellular stress to search for the functional relationships between stress granules and TG2. Using immunofluorescence staining, we found that some substrates of TG2 are localized in stress granules. These include APG-1, Tom34 and PDIA4, all of which are novel components of stress granules. We therefore propose that TG2 could assist the formation of stress granules. However, in vivo and in vitro transamidation experiments showed that arsenic trioxide decreased the catalytic activity of TG2 by reacting with its thiol group, ruling out the possibility of the catalytic role of TG2 in the formation of stress granules. The results suggest that the decreased activity of TG2 could explain for the therapeutic mechanisms of arsenic trioxide in treating PML. Additionally, when Cos-1 cells were treated with arsenic trioxide, TG2 seemed to co-localize with the structure of mitochondria, surrounding the nucleus and the microtubule-organizing center (MTOC). We also detected an increase in the expression of Mitofusin-1 and Mitofusin-2, suggesting the formation of mitochondrial fusion. This phenomenon coincides with increased reactive oxygen species, decreased mitochondrial membrane potential. Furthermore, we also detected apoptosis through the mitochondrial release of cytochrome c. The results provide a new strategy to study the relationship between apoptosis and mitochondrial fusion. Co-immunoprecipitation experiments also demonstrated a strong binding affinity between TG2 and Grp78, implying that Grp78 could be a chaperone for TG2 under conditions of oxidative stress. These observations are interesting, but at this point, we know little about the roles played by TG2 in the formation of mitochondrial fusion.中文摘要: i文摘要: ii寫表: iv錄: vi一章 前言 1.1砷化物的介紹 1.1.1三氧化二砷的應用與造成的影響 1.1.2三氧化二砷在細胞內的分子機制 2.1.3三氧化二砷與細胞內的壓力顆粒(Stress Granule：SG) 4.2 轉穀氨醯胺酶II(transglutaminse II：TG2)的介紹 9.3 研究動機 11二章 實驗材料與方法 15.1細胞株培養 15.1.1繼代培養 15.1.2細胞冷凍與解凍 15.2藥物配製與加藥處理 15.3 反轉錄聚合酶連鎖反應(RT-PCR) 15.3.1抽RNA 16.3.2反轉錄反應(reverse transcription) 16.3.3聚合酶連鎖反應((Polymerase Chain Reaction：PCR) 17.3.4洋菜膠電泳 17.4. 蛋白質分析法 18.4.1蛋白質萃取 18.4.2 Tricine/SDS聚丙烯醯胺凝膠電泳(Tricine/SDS-PAGE) (79) 18.4.3西方墨點法(western blotting) 19.4.4 蛋白質膠片染色(protein coomassie blue G-250 stainning) 20.4.5 銀染色法(silver staining) 20.5免疫螢光染色法(immunoflurorescence： IF) 20.6 共同免疫沉澱反應(Co-Immunoprecipitation：CO-IP) 21.7胞內鈣離子的測定 21.8粒線體膜電位的測定 22.8.1粒線體蛋白的抽取 22.9細胞內活性氧分子(ROS: reactive oxygen species)的測定 22.10 製備多株抗體 22.10.1 核酸引子的設計 22.10.2核酸酵素切割與片段純化 23.10.3重組質體的接合(Ligation)及轉形作用(Transformation) 23.10.4 純化重組蛋白 24.10.5 抗原的製備 24.10.6多株抗體血清的製備 24.11 in vivo transamidation 25.12 in vitro transamidation 25三章 結果 26.1 TG2部分受質的多株抗體製備 26.2 三氧化二砷引起Cos-1細胞株壓力顆粒的產生 26.3 壓力顆粒與TG2和TG2部分受質的關係 27.4 三氧化二砷處理下，細胞內鈣離子與TG2活性，RNA及蛋白質表現量的影響 27.5 三氧化二砷處理下，在細胞內TG2分佈位置與胞器之間的關係 29.6 三氧化二砷會造成Cos-1細胞產生mitoptosis 30.7 三氧化二砷所造成的mitoptosis與mitochondrial fusion之間的關係 30.8 三氧化二砷對TG2與內質網chaperone Grp78的結合關係 32.9 三氧化二砷與Grp78和Bcl-2之間的關係 32四章 討論與總結 34.1 三氧化二砷利用砷攻擊TG2上的thiol group (SH)造成細胞內TG2的活性下降 34.2三氧化二砷造成粒線體聚集在細胞核和MTOC周邊只發生粒線體融合反應(mitochondrial fusion)不發生粒線體分裂反應(fission) 35.3 三氧化二砷造成TG2座落在粒線體結構中可能扮演的角色 37.4 SGs新成分的發現 38.5 總結 39五章 實驗結果圖表 40考文獻： 58application/pdf2054765 bytesapplication/pdfen-US壓力顆粒活性氧自由基粒線體膜電位轉穀氨醯胺酶粒線體融合反應三氧化二砷stress granulereactive oxygen speciesmitochondrial membrane potentialmitochondrial fusiontransglutaminase IIarsenic trioxide[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178909/1/ntu-98-R96b46006-1.pdf