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

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。

關鍵字

第二型轉麩胺酶細胞凋亡；鈣離子平衡

並列摘要

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.

並列關鍵字

TG2 ； apoptosis ； caclium homeostasis

參考文獻

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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

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