Abstract
摘要:ATP合成酶是細胞中普遍存在的蛋白質複合體,它能產生「能量貨幣分子」—ATP。這個分子裝置含有兩個部分,穿膜的部分(Fo)會因質子梯度而旋轉,以及催化產生ATP的部分(F1)。ATP 合成酶一般被認為出現在粒線體內膜上,然而愈來愈多的證據顯示ATP合成酶也會表現在癌組織的表皮細胞、乳癌及肺癌細胞的細胞膜上,這一類的ATP 合成酶被稱為「細胞膜異位表達ATP合成酶」。除了我們先前一連串的研究發現之外,有許多其他學者的研究也證明阻斷細胞膜異位表達ATP合成酶的活性可以有效地抑制癌細胞生長,因而推論細胞膜異位表達ATP合成酶具有可當作抗腫瘤及抑制血管新生藥物標靶的潛力。然而,這些癌細胞是如何引導ATP合成酶移動到細胞表面仍然不清楚。在粒線體和細胞膜上的ATP合成酶結構是否不同呢? 需要更進一步的研究。後轉譯修飾作用(例如磷酸化和乙醯化)在蛋白質運送過程扮演重要的角色,那麼蛋白質的磷酸化和乙醯化是如何調節ATP合成酶的運送呢? 這仍是一個未解的問題。因此本計畫的主要目標是要以蛋白體學技術探討受異位表達ATP合成酶運送影響之磷酸化與乙醯化交互作用動態變化。
特定目標:
1. 以低温電子顯微鏡釐清細胞膜上的ATP合成酶結構是否與粒線體不同。
2. 剖析ATP合成酶從粒線體運送至細胞膜的路徑。
3. 解析受關鍵性運送蛋白質所影響的蛋白體及膜蛋白體。
4. 以磷酸化蛋白體學闡明參與ATP合成酶運送的磷酸化蛋白質。
5. 以乙醯化蛋白體學鑑定參與ATP合成酶運送的乙醯化蛋白質。
6. 探討受異位表達ATP合成酶運送影響之磷酸化與乙醯化交互作用動態變化。
在本研究計畫中,我們期望能夠了解在細胞膜及粒線體上的ATP合成酶結構,以及它如何從粒線體運送至細胞膜的路徑;除此之外,我們將能找到參與此運送過程的重要磷酸化和乙醯化蛋白質,進而釐清它們的交互作用動態變化,這將有助於抗癌治療策略的發展。
Abstract: ATP synthase is a ubiquitous multimeric protein complex that catalyzes the synthesis of ATP, the common “energy currency” of living cells. This molecular machine consists of two moieties, a transmembrane portion (Fo), the rotation of which is induced by the proton gradient, and a globular catalytic moiety (F1) that synthesizes ATP. In general, it is localized to the mitochondrial inner membrane. Recent studies showed that ATP synthase was also found on the extracellular surface of endothelial cells in some cancer tissues, lymphocytes, hepatocytes, paraganglioma, proliferating cell lines, breast and lung cancer cells. With the property of facing out-side the cell, this kind of ATP synthase is called ectopic ATP synthase. Ectopic ATP synthase is expressed on the surfaces of various cancer cells, but not on the normal or normal-like cells, therefore, the researchers suggest that ectopic ATP synthase is a potential molecular target for anti-tumor and anti-angiogenesis therapies. Inhibition of ectopic ATP synthase showed inhibitory effects on cell proliferation in various cancer cells, suggesting the oncogenic role of ectopic ATP synthase in tumorigenesis. However, how the cancer cells direct ATP synthase towards cell surface remains unclear. Whether the ATP synthase structures on mitochondria and plasma membrane are different require further investigation. Post-translational modifications such as phosphorylation and acetylation play important roles in regulating protein trafficking. However, how the protein phosphorylation and acetylation regulate the ATP synthase trafficking is still uncertain. Our major objective is to elucidate the interplay of phosphorylation and acetylation dynamics in ectopic ATP synthase trafficking by proteomics approaches. Our specific aims are:
1. To explore whether the complex structures of ATP synthase on plasma membrane are different from mitochondria by cryo-electron microscopy (cryo-EM).
2. To reveal the trafficking pathways of ATP synthase from mitochondria to plasma membrane.
3. To profile the global and membrane proteomes affected by the key trafficking proteins.
4. To elucidate the involved phosphoproteins in ATP synthase trafficking using phosphoproteomics.
5. To identify the key acetylated proteins involved in ATP synthase trafficking using acetylomics.
6. To study the interplay of phosphorylation and acetylation dynamics in ectopic ATP synthase trafficking.
With the proposed study, we expect to understand the ATP synthase structures on mitochondria and plasma membrane as well as the trafficking pathways of ectopic ATP synthase from mitochondria to cell surface. In addition, we will further elucidate the involved phosphoproteins and acetylated proteins in ATP synthase trafficking. The interplay of phosphorylation and acetylation dynamics in the trafficking pathways could be useful in the development of therapeutic strategies against cancers in which signaling are perturbed.
Keyword(s)
細胞膜異位表達ATP合成酶
蛋白體學
磷酸化蛋白體學
乙醯化蛋白體學
網路動態
ectopic ATP synthase
proteomics
phosphoproteomics
acetylomics
network dynamics