ATP合成酶異位表現至肺癌細胞表面之運輸途徑

Abstract

ATP合成酶是一種以多個次單元組合而成的蛋白複合體，可以進行ATP的生合成，並提供細胞生存所需的能量。粒線體是細胞中重要的ATP合成胞器，因此，長久以來ATP合成酶被認為僅在粒線體中表現；然而，一些近期的研究發現ATP合成酶在癌組織的表皮細胞、淋巴細胞、肝細胞，以及乳癌和肺癌細胞株的細胞膜表現，這一類ATP合成酶我們稱它們為「異位表達ATP合成酶」。這些ATP合成酶具有在胞外合成ATP的功能，我們先前的研究發現透過藥物citreoviridin可以抑制其ATP產生的活性，進而引發癌細胞的死亡，然而它們是如何被運輸至細胞表面的機制至今仍然不清楚。為了找出參與在這種運輸機制的蛋白分子，我們使用RNA干擾為基礎的篩選實驗，首先在抑制25個參與運送有關的基因表現後，以細胞ELISA測量細胞表面的ATP合成酶表現量；我們同時也以流式細胞儀和細胞免疫螢光染色方法，驗證在細胞ELISA的實驗中被認為涉及ATP合成酶運輸的蛋白，是否真的可以改變細胞膜上ATP合成酶的表現量。結果發現有PARK2、MFN1以及COPA等基因可能在異位表現之ATP合成酶組裝、運輸機制中，扮演重要的角色。

ATP synthase is a multimeric protein complex that catalyzes the synthesis of ATP. For a long time, animal ATP synthase was believed to be found only in mitochondria, where most cellular ATP synthesis takes place. However, in recent studies ATP synthase was also found on the extracellular surface of endothelial cells in some cancer tissues, lymphocytes, hepatocytes, proliferating cell lines, breast cancer and lung cancer cells. With the property of facing out-side the cell, this kind of ATP synthase is called ectopic ATP synthase. Our previous studies found that treating lung and breast cells with drug such as citreoviridin inhibited ectopic ATP synthase and caused the cancer cell death. However, how ATP synthase expressed on plasma membrane is still unclear. Therefore, to reveal what kind of the molecules are involved in the ectopic expression of ATP synthase, the RNA interference screening in lung cancer A549 cells was performed. Twenty-five genes were silenced, and then ectopic ATP synthase expressions were measured by cell ELISA assay. Several genes are potentially involved in ectopic ATP synthase expression. The alternations of ectopic ATP synthase expression were further confirmed by flow cytometry and immunocytochemistry. These results suggest that PARK2, MFN1 and COPA may play crucial roles in the trafficking and assembling mechanism of ectopic ATP synthase.