Abstract
摘要:在所有多細胞生物中細胞與細胞間的溝通是必要的。細胞可藉由分泌一些影響其鄰近細胞或是改變細胞外生化特性的因子以反應外界環境的改變。有許多的研究認為這是由胞外囊泡所調節的。胞外囊泡會傳送許多不同的生物分子給受體細胞,包括DNA、RNA、蛋白質和脂質,最近它被認為是一種很普遍存在的細胞間溝通方式。癌細胞會傳送帶有致癌基因或是蛋白質的胞外囊泡,而促使癌症形成。因此胞外囊泡的研究成為癌症研究中極為基本且有趣的一環。胞外囊泡的釋放和攝取是需要能量的過程,而活細胞中最常用的能量貨幣是由ATP合成酶所產生的ATP。ATP合成酶不僅出現在粒線體內膜上,同時也在細胞膜上,尤其是在小窩和脂筏。很有趣地,研究者發現胞外囊泡的攝取路徑包括了網格蛋白依賴及非依賴的內吞作用,例如微囊蛋白調節的攝取和脂筏調節的內化作用。除此之外,越來越多的證據指出胞外囊泡分泌和攝取是由特殊的蛋白質間交互作用所調節。最近,蛋白體學技術常被用於尋找特殊胞外囊泡的標記分子,其研究顯示許多胞外囊泡和微泡中都有ATP合成酶次單元體存在。近十年來的研究顯示,在生理壓力下,胞外囊泡常被用來調節細胞間的溝通,因此使學者們提高對於研究它的興趣。缺氧和饑餓是兩種與癌症進程高度相關的壓力,它們會調節胞外囊泡的釋放和運送,並且會造成ATP釋放至胞外。然而,仍有許多不清楚且未解的問題。我們在此計畫中提出了假說,在細胞表面的ATP合成酶會產生ATP到細胞外而影響胞外囊泡的攝取和釋放,在胞外囊泡膜上的蛋白質會和ATP合成酶交互作用而影響這個過程。除此之外,ATP合成酶會和胞外囊泡的蛋白質交互作用而參與細胞與細胞間的溝通。因此,本計畫主要目標是要以蛋白體學探討ATP合成酶在藉由胞外囊泡而進行細胞間溝通所扮演的角色。
特定目標:
1. 確定在缺氧和饑餓壓力下ATP合成酶是否會釋放到胞外囊泡。
2. 解析在缺氧和饑餓壓力下胞外囊泡的蛋白體。
3. 鑑定在胞外囊泡中與ATP合成酶交互作用的蛋白質。
4. 找出與細胞表面ATP合成酶交互作用的胞外囊泡膜蛋白質。
5. 闡明被ATP合成酶調節之胞外囊泡參與的細胞間溝通。
6. 檢視異位表達ATP合成酶和它的交互作用蛋白質是否會影響受體細胞攝取胞外囊泡。
在本研究計畫中,我們期望能夠了解ATP合成酶調控的胞外囊泡運送之分子機制。除此之外,我們將進一步釐清在胞外囊泡釋放和攝取中,異位表達ATP合成酶的交互作用蛋白質所扮演的功能。我們將能獲知異位表達ATP合成酶透過胞外囊泡所調控的細胞間溝通之角色,進一步透過干擾胞外囊泡運送路徑而提供有用的治療策略。
Abstract: Cell–cell communication is essential for all multicellular organisms. Cells response to their local environments by secreting factors which either affect neighboring cells or change the biochemical properties of extracellular space. Intensive investigation suggests that these events are mediated by extracellular vesicles (EVs). EVs transfer many types of biomolecules including DNA, RNA, proteins and lipids to their recipient cells and are recently recognized as a general mode of cell-cell communication. Cancer cells transfer EVs to carry oncogenes, or oncogenic proteins that promote cancer formation. Therefore, EVs are becoming an appreciated fundamental and interesting aspect in cancer research. Both EV uptake of recipient cells and release by donor cells are energy-requiring processes. The common “energy currency” of living cells is ATP, produced by ATP synthase. ATP synthase was found not only on mitochondrial inner membrane but also on plasma membrane, especially in caveolae and lipid raft. Interestingly, EV uptake pathways include clathrin-dependent and independent endocytosis such as caveolin-mediated uptake and lipid raft-mediated internalization. Moreover, a growing accumulation of evidence indicates that EV secretion and uptake are mediated by specific protein–protein interactions. Recently, proteomics approach becomes a very useful and promising technique to identify specific markers in EVs. Proteomic studies reported that many ATP synthase subunits were identified in exosomes and shed microvesicles. The last decade has seen a rapid expansion of interest in EVs, proposed to mediate cell–cell communication in physiological conditions such as stress. Hypoxia and starvation, two kinds of stress highly related to cancer progression, mediated secretion and trafficking of EVs in tumor progression and caused the release of ATP to extracellular space. However, there are still many unclear and unsolved problems. Here, we proposed a hypothesis that ATP synthase on the cell surface may produce extracellular ATP for EV uptake and release, the proteins on EV membrane may interact with ATP synthase for the uptake and release processes. Additionally, ATP synthase interact with the proteins of EVs to get involved in cell-cell communications. Therefore, our major objective is to elucidate the role of ectopic ATP synthase in cell-to-cell communication by EVs. Our specific aims are:
1. To determine whether ATP synthase complexes are released into EVs under hypoxia or serum deprivation.
2. To profile the proteome of EVs under hypoxia or serum deprivation.
3. To identify the proteins interacting with ATP synthase in EVs.
4. To reveal the proteins on EV membrane interacting with ATP synthase on cell surface.
5. To elucidate cell-to-cell communication via EVs by ectopic ATP synthase.
6. To examine whether ectopic ATP synthase and its interacting proteins affect EV uptake by recipient cells.
With the proposed study, we expect to understand the molecular mechanisms involved in the ATP synthase-mediated EV transfer. In addition, we will further elucidate the functions of ectopic ATP synthase-interacting proteins in EV release and uptake. This proposed research will provide in-depth information on the role of ectopic ATP synthase in cell-cell communications mediated by EVs and useful therapeutic strategies against cancers in which EV transfer pathways are perturbed.
Keyword(s)
胞外囊泡
細胞與細胞間的溝通
ATP合成酶
蛋白體學
壓力
extracellular vesicles
cell–cell communication
ATP synthase
proteomics
stress