探討Cos-1細胞在三氧化二砷所引起的氧化壓力下轉穀氨醯胺酶II及它的受質的角色

Abstract

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.