異位表達ATP合成酶經由KIF5B與Drp1交互作用的運輸途徑探討

Abstract

三磷酸腺苷合成酶(ATP synthase)位於粒線體內膜上，是一種負責產生三磷酸腺苷(ATP)並供給細胞內多種反應途徑使用的酵素。我們先前的研究指出三磷酸腺苷合成酶會出現在許多不同種類癌細胞的細胞膜上，稱為異位表達三磷酸腺苷合成酶(ectopic ATP synthase)；然而，關於異位表達三磷酸腺苷合成酶在細胞內的運輸路徑還不是非常清楚。為了進一步研究三磷酸腺苷合成酶參與在運輸路徑中的狀況，根據先前基因集富集分析的結果，我們推測三磷酸腺苷合成酶是以完整複合體的形式存在於粒線體內部，隨著粒線體沿著細胞骨架被運輸到細胞膜上，進而也被運送至細胞膜。為了證實這項推測，我們在神經細胞SK-N-BE(2)C加入抑制細胞骨架聚合的藥物-諾考達唑(nocodazole)，加藥後不管是免疫螢光染色與流式細胞儀實驗結果都顯示異位表達三磷酸腺苷合成酶的表現量下降。此外將驅動蛋白5B(Kinesin family member 5B,KIF5B) 經由小分子干擾核糖核酸（small interfering RNA）抑制其基因表現後，在抑制免疫螢光染色與流式細胞儀實驗，兩者的實驗結果中異位表達三磷酸腺苷合成酶的表現量下降。另一方面我們也藉由小分子干擾核糖核酸去抑制會造成粒線體的碎裂化(Fission)的動力激活蛋白(Dynamin-1-like protein, Drp1) 與利用質體在細胞中過度表現 Drp1 野生株、其C 端 或N端，在免疫螢光染色與流式細胞儀實驗看到異位表達三磷酸腺苷合成酶的表現量與DRP1 表現量成正相關，尤其Drp1 C 端的影響較N 端顯著。此外我們也利用網路資料庫與蛋白質-蛋白質對接(protein-protein docking)網路工具去預測驅動蛋白與動力激活蛋白是否直接相連。根據上述的實驗結果，我們推測 KIF5B-Drp複合體連結粒線體與細胞骨架的運輸模式，會在異位表達三磷酸腺苷合成酶的運輸途徑當中扮演重要角色。

Adenosine triphosphate (ATP) synthase, an inner membrane enzyme of mitochondria, is essential for ATP production in many cell biological processes. Our previous studies have shown that ATP synthases not only existed on mitochondrial inner membrane but also plasma membrane (ectopic ATP synthases) in several cancer cell lines. However, the trafficking mechanism of ATP synthase to cell surface is still required further investigation. According to our previous gene set enrichment analysis (GSEA) results, we inferred ectopic ATP synthases transported to cell surface through the microtubule-mediated mitochondria trafficking. To examine whether this presumption is correct, we conducted flow cytometry and immunocytochemistry (ICC) after treating nocodazole, a microtubule-depolymerizing agent, in cancer cells. The results revealed that microtubule disruption reduced ectopic ATP synthase expression level. In addition, silencing kinesin family member 5B (KIF5B), a microtubule motor protein, with small interfering RNA showed the similar trend with the results of microtubule disruption. On the other hand, we also found that mitochondria dynamic related to ectopic ATP synthase expression. Both flow cytometry and ICC demonstrated that mitochondrial fission protein, dynamic-related protein 1 (Drp1), knockdown and overexpression resulted in low and high ectopic ATP synthase expression respectively. In addition, Drp1 C-terminus was showed more significant than N-terminus in ectopic ATP synthase expression in overexpression experiments. Moreover, we used protein-protein interaction database and docking web server to predict whether KIF5B bound with Drp1 directly. Taken together, these findings suggest that KIF5B-Drp1 complex-mediated mitochondrial trafficking via microtubule may play a crucial role in ectopic ATP synthases transport.