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研究生:謝雨帆
研究生(外文):Yu-Fan
論文名稱:第二型轉麩胺酶藉由其受質RAP1GDS1參與調節細胞內鈣離子的平衡
論文名稱(外文):RAP1GDS1 is the substrate of TG2 for modulating intracellular calcium homeostasis
指導教授:蔡嘉哲蔡嘉哲引用關係
學位類別:博士
校院名稱:中山醫學大學
系所名稱:微生物免疫研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:122
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背景:第二型轉麩胺酶(Type 2 transglutaminase, TG2)是一種多功能蛋白,能夠仰賴鈣離子的催化進行蛋白質後修飾作用,此外,具有G蛋白的功能進行訊息傳遞,細胞表面的功能則與細胞貼附有關,TG2也曾被發現能分佈在粒線體參與細胞凋亡過程。調控細胞內質網和粒線體間鈣離子的流動與誘導粒線體死亡路徑有關,但是,TG2如何參與調控其中的機制仍不清楚,因此,我們的研究在於了解TG2的功能是如何參與T細胞的凋亡及細胞內鈣離子調控。
材料方法:我們利用人類急性淋巴瘤細胞株(Jurkat cell)轉染野生型和不具活性之突變型TG2質體,建立穩定的條件式表達系統 (Tet-on system)細胞株。以TG2缺陷的老鼠胚胎纖維母細胞 (mouse embryonic fibroblasts, MEFs)作為對照,研究TG2與鈣離子調控的關係。Tet-On cells以Doxycycline處理使之大量表現TG2,檢測大量表達TG2之細胞株細胞凋亡的情形,包括,以流式細胞儀分析sub-G1的比例,DNA片段化的情形和粒線體膜電位的變化。利用各種細胞胞器螢光染劑分別檢測細胞內細胞質、粒線體和內質網內鈣離子的變化。使用蛋白質體學鑑定大量表現TG2細胞株蛋白質的變化,找出可能參與細胞內鈣離子調控之分子。
結果:大量誘導野生型的TG2細胞株的死亡情形比不具轉麩胺酶活性的突變型細胞株嚴重,同時伴隨著大量粒線體鈣離子堆積和內質網鈣離子的釋放。在投與SERCA幫浦抑制劑後(thapsigargin),可觀察到短時間野生型細胞株鈣離子堆積的幅度大於野生型細胞株。相反的,檢測TG2 KO MEF則發現,粒線體鈣離子吸收和內質網鈣離子的釋放釋放程度降低,細胞質內鈣離子則無明顯的差別。抑制轉麩胺酶活性或內質網鈣離子通道則會降低粒線體鈣離子的吸收。由蛋白質體學分析鑑定分析後發現RAP1GDS1為TG2的受質。以RAP1GDS1 shRNA抑制其蛋白質的表現會降低TG2所誘導之線體鈣離子的吸收和內質網鈣離子的釋放。
結論: 轉麩胺酶活性與細胞凋亡有關且能造成粒線體鈣離子的吸收和內質網鈣離子的釋放。TG2 藉由其受質RAP1GDS1參與TG2所誘導之細胞內鈣離子的平衡。


Background: Type 2 transglutaminase (TG2) is a multifunctional protein, which catalyzes Ca2+-dependent protein modifications, acts as a G protein in transmembrane signaling and cell surface adhesion mediator. It not only has a pro-apoptotic function but also presences in the mitochondria and participates in the molecular events of apoptosis. Modulating calcium signaling from the endoplasmic reticulum (ER) to mitochondria can be critical in the induction of mitochondrial dependent cell death pathway; however, the mechanism of action of TG2 is unknown. Accordingly, we investigate the effect of TG2 in calcium signaling pathway which are known to regulate apoptosis.
Materials and methods: Jurkat cells were transfected with human wild type TG2 (wtTG2) and TG2C277S which is the transamidation activity mutant by a tetracycline-induced system. Mouse embryonic fibroblasts deficient for TG2 (TG2 KO MEF) were used for calcium experiment. Apoptotsis was determined by DNA fragmentation and flow cytometry. The levels of mitochondria, cytosolic and ER calcium were determined in cells staining with Rhod-2-AM, Fura-2 and Mag-Fura 2, respectively. TMB-8 and ryanodine were used to determine TG2 participation of the inositol trisphosphate receptor and ryanodine-sensitive receptor mediated calcium release from the ER, respectively. Two-dimensional gel electrophoresis (2-DE) was used to analyze proteins on the TG2 overexpressing cells.
Results: Overexpression of wtTG2 induced Jurkat cells apoptosis. TG2 was found to be colocalized with calcium in mitochondria. Overexpression of wtTG2 in Jurkat cell has more mitochondrial calcium accumulation. Mitochondrial calcium uptake and ER calcium release were enhanced immediately in wtTG2 cells evoked by thapsigargin (tg). Using TG2 KO MEF cells evoked by tg, mitochondrial calcium uptake and ER calcium release were decreased. Treatment of cells with TMB-8 or Ryanodine resulted in suppressed the elevation of mitochondrial calcium uptake in cells evoked by tg. After 2-DE analysis, RAP1GDS1 became a candidate of intracellular calcium signaling regulated protein. RAP1GDS1 depletion suppressed TG2 modulating mitochondrial calcium uptake and ER calcium release in Tet-on wtTG2 cells evoked by tg.
Conclusions: The transamidation activity of TG2 increase mitochondrial calcium uptake mediated calcium release from ER, which contribute to cell death. RAP1GDS1 is the substrate of TG2 and influences TG2 modulating intracellular calcium homeostasis.



目錄
中文摘要 i
英文摘要(Abstract) ii
第一章 緒論 1
第一節 細胞凋亡與細胞凋亡之重要性 1
第二節 細胞凋亡訊息傳遞路徑 2
第三節 參與細胞凋亡之分子 4
第四節 粒線體與細胞凋亡 6
第五節 細胞內之鈣離子調控與細胞凋亡 9
第六節 第二型轉麩胺酶 (Transglutaminase 2) 13
第七節 第二型轉麩胺酶與細胞凋亡 18
第八節 研究的動機與目的 22
第二章 研究方法與材料 24
一、材料及藥品 24
二、細胞培養 24
三、細胞存活率(viability)分析 25
四、細胞存活率分析(MTT assay) 25
五、質體構築 26
六、細胞轉染(transfection) 28
七、共軛焦顯微鏡觀察(Confocol microscopy) 28
八、細胞凋亡DNA片段化實驗 29
九、流式細胞儀分析細胞凋亡情形 30
十、流式細胞儀分析細胞粒線體膜電位 30
十一、RNA萃取 31
十二、反轉錄聚合酶反應 (Reverse transcription-polymerase chain reaction; RT-PCR) 32
十三、洋菜膠電泳(Agarose gel) 33
十四、製備粒線體蛋白溶液 33
十五、細胞進行聚丙烯醯胺膠體電泳之前處理 34
十六、西方墨點法 (western blot) 34
十七、測量粒線體中鈣離子 35
十八、測量細胞內細胞質鈣離子 35
十九、測量細胞內質網鈣離子 36
二十、轉麩胺酶酵素活性測量之細胞溶液前處理 36
二十一、轉麩胺酶酵素活性測量 37
二十二、二維電泳分析(2-dimensional analysis) 37
二十三、統計分析 39
第三章 結果 40
第一部份 建立條件式誘導基因表現實驗模組 (conditional expression model)-Tetracyclin inducible gene expression system (Tet-On system) 40
第二部份 誘導TG2蛋白質大量表現引發Tet-On wtTG2和Tet-On TG2C277S細胞株產生細胞凋亡 42
(一) 誘導TG2蛋白質大量表現引發生長抑制和細胞死亡 42
(二) 誘導TG2蛋白質大量表現引發Tet-On wtTG2和Tet-On TG2C277S細胞株產生細胞凋亡 42
(三) 誘導TG2蛋白質大量表現引發Tet-On wtTG2和Tet-On TG2C277S細胞凋亡現象是經由粒線體死亡路徑 44
第三部份 TG2蛋白質對細胞內鈣離子的影響 46
(一) 誘導TG2蛋白質大量表現引發TG2轉位至粒線體且TG2蛋白質的分布與粒線體鈣離子堆積的位置相同 46
(二) 誘導Tet-On wtTG2和Tet-On TG2C277S細胞株大量表現TG2對細胞內鈣離子的影響 47
(三) TG2缺陷之老鼠胚胎纖維母細胞(TG2 KO MEF)對細胞內鈣離子的影響 51
(四)抑制內質網鈣離子釋放通道對TG2誘導粒線體鈣離子堆積的影響 52
(五) 轉麩胺酶活性對TG2所參與調控粒線體鈣離子吸收之影響 53
第四部分 TG2蛋白質如何參與細胞內鈣離子調控 55
(一) 以蛋白質體學方法鑑定大量表現TG2之Tet-On wtTG2細胞株所影響之蛋白質表現量 55
(二) 研究RAP1GDS1參與TG2調控之鈣離子平衡之可能 56
第四章 討論 60
第一部份 建立條件式誘導基因表現實驗模組 (conditional expression model)-Tet-On system 60
第二部份 誘導TG2蛋白質大量表現引發Tet-On wtTG2和Tet-On TG2C277S細胞株產生細胞凋亡 61
第三部份 TG2蛋白質對細胞內鈣離子的影響 66
第四部分 TG2蛋白質如何參與細胞內鈣離子調控 74
第五部分 結論 77
第六部分 展望與應用 78
第五章 附 錄 80
圖一、TG2 functional element 80
圖二、TG2酵素催化反應 81
圖三、Tet-on系統介紹 82
第六章 圖與表 84
第七章 參考文獻 103
圖目錄
圖一、建立條件式誘導基因表現實驗模組 (conditional expression model)-Tetracyclin inducible gene expression system (Tet-On system)。 83
圖二、誘導TG2蛋白質大量表現引發Tet-On wtTG2和Tet-On TG2C277S細胞產生細胞凋亡現象。 85
圖三、誘導TG2蛋白質大量表現引發TG2轉位至粒線體且TG2蛋白質的分佈與粒線體鈣離子堆積的位置相同。 86
圖四、誘導Tet-On wtTG2大量表現TG2蛋白質增強粒線體鈣離子的堆積和內質網鈣離子的釋放。 87
圖五、TG2缺陷之老鼠胚胎纖維母細胞(mouse embryonic fibroblast)降低tg誘導之粒線體鈣離子吸收和內質網鈣離子的釋放。 89
圖六、鈣離子鰲合劑TMB-8和Ryanodine抑制Tet-On wtTG2和Tet-On TG2C277S細胞株粒線體鈣離子的吸收。 90
圖七、鈣離子鰲合劑TMB-8和Ryanodine抑制野生型老鼠胚胎纖維母細胞(WT MEF)粒線體鈣離子的吸收。 91
圖八、轉麩胺酶活性抑制劑ZDON對TG2缺陷之老鼠胚胎纖維母細胞粒線體鈣離子的影響。 92
圖九、Tet-On vector細胞之二維電泳分析蛋白質表現量。 93
圖十、Tet-on wtTG2細胞之二維電泳分析蛋白質表現量。 94
圖十一、Tet-on TG2C277S細胞之二維電泳分析蛋白質表現量。 95
圖十二、西方墨點法檢測RAP1GDS1蛋白質的表現。 96
圖十三、檢測knock down Tet-on wtTG2細胞株之RAP1GDS1後其蛋白質表現。 97
圖十四、knock down Tet-On wtTG2之RAP1GDS1抑制粒線體鈣離子的堆積和內質網鈣離子的釋放。 98
圖十五、TG2蛋白質參與細胞凋亡和細胞內鈣離子調控之實驗假說。 99

表目錄
表一、與Tet-on TGC277S細胞相比較,Tet-on wtTG2細胞株經二維電泳分析後以質譜(LC-MS/MS)分析鑑定表現量有差異之蛋白質。 101



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