臺灣博碩士論文加值系統

異位表達ATP合成酶（ectopic ATP synthase）在許多癌症細胞中被發現會在細胞膜表面合成胞外ATP。近來許多研究透過蛋白質體技術，發現異位表達ATP合成酶會出現在胞外囊泡（extracellular vesicles）中，且在細胞與細胞間的溝通中扮演重要角色，然而其功能仍未知。我們將肺腺癌細胞A549培養在含有0.1% 胎牛血清的細胞培養液中，藉此增加細胞膜上的異位表達ATP合成酶，我們使用梯度離心分離出培養液中的胞外囊泡，包含微泡（microvesicles）及外泌體（exosomes），並且利用穿透式電子顯微鏡、奈米粒子追蹤分析儀及囊泡特定的蛋白質標記確認離心的結果。我們也利用西方墨點法確認異位表達的ATP合成酶是否存在於胞外囊泡中，發現從低血清的培養液分離出的外泌體數量較多且其中的異位表達ATP合成酶表現量較高。為了進一步確認其功能，我們首先測試外泌體中的異位表達ATP合成酶是否能製造胞外ATP，根據ATP冷光探測實驗的結果是不會產出ATP至培養液中。為了瞭解外泌體中的異位表達ATP合成酶的功能，我們接下來分離正常血清濃度及低血清濃度培養液的微泡及外泌體進行蛋白體學實驗，並以質譜鑑定其蛋白身分。根據基因集富集分析(gene set enrichment analysis, GSEA)的結果，我們發現除了代謝及細胞凋亡的功能會顯著增加外，與免疫有關路徑的功能也會增加，因此我們認為低血清濃度的外泌體可能具有免疫相關的作用。我們進一步將A549的外泌體和人類淋巴球Jurkat T 細胞共培養，發現低血清濃度分離的外泌體會讓Jurkat細胞存活率大幅下降，而經過ATP合成酶抑制藥物黃毒素(citreoviridin)作用的外泌體則是不會對Jurkat細胞的生長造成影響，因此我們猜測這可能和異位表達的ATP合成酶有關。為了瞭解外泌體中的異位表達ATP合成酶是如何影響Jurkat T細胞，我們透過交互檢視之前的人類蛋白質體晶片資料與已發表期刊論文中的Jurkat T細胞蛋白質體資料，找出外泌體中的異味表達ATP合成酶會與Jurkat T細胞中的哪些蛋白有交互作用，最終我們找到了FYN蛋白質，一種在Jurkat T細胞表面的活性酶，且和T細胞受體(T cell receptor)的訊號傳遞有關，我們推測異位表達ATP合成酶會和FYN交互作用，進而影響Jurkat T細胞的生長，因此我們利用分子對接(Docking)的軟體，預測出ATP合成酶有很大的機會會和FYN對接，而利用ATP合成酶的抗體阻斷胞外囊泡的ATP合成酶的實驗顯示，Jurkat T細胞的生長的確和胞外囊泡中的ATP合成酶有關，反之，我們利用FYN抗體阻斷Jurkat T細胞上的FYN，即使加了低血清濃度分離的外泌體也不會對Jurkat T細胞的生長造成影響，但詳細的機制仍需要進一步實驗證明。根據上述的結果，我們認為血清缺乏後，外泌體中的異位表達ATP合成酶會增加，而且會和人類淋巴球Jurkat T 細胞表面的FYN蛋白作用，進而去影響其生長。

Ectopic adenosine triphosphate synthase (eATP synthase) was found on the cell surface to generate extracellular ATP in various cancer cells. Recently using proteomics approach, several reports identified ATP synthase in extracellular vesicles (EVs) which are important in cell-cell communications. However, the functions of ATP synthase in EVs are still unclear. Here we increased the expression of eATP synthase on the plasma membrane of lung cancer A549 cells via 0.1% FBS starvation and collected EVs including microvesicles and exosomes from the culture medium using serial centrifugation. Both of them were assuredly isolated and confirmed by transmission electron microscopy, nano tracking analysis, and specific EV markers. We also used western blot to demonstrate the presence of ATP synthase in EVs. Besides, dot blot experiment revealed that the F1 portion faced towards extracellular space using anti-ATP beta subunit antibody. Under starvation, the expression of eATP synthase was enhanced on cell surface and exosomes as well as increased EV secretion. To further investigate the functions, we first elucidated whether eATP synthase on EV membrane produced ATP by ATP luciferase analysis. Our data showed that there was no significant ATP production. Moreover, we performed proteomic analysis using dimethyl labeling and LC-MS/MS to compare the difference between EVs released by A549 control and starvation. We identified a total of 1504 and 869 proteins in microvesicles and exosomes, respectively. Using 1.5- and 0.67-fold change as the thresholds, there were 171 and 46 differential expressed proteins in microvesicles and exosomes, respectively. Seven ATP synthase subunits in microvesicles and five in exosomes were identified. The proteomic data were further analyzed using gene set enrichment analysis (GSEA) within the C2 (curated gene sets) subclasses and setting 0.25 as FDR cutoff to find out biological functions related to eATP synthase. Among these up-regulated terms, metabolism, cell cycle and apoptosis were enriched, which indicating that starvation influences cell behavior. Additionally, the immune response is upregulated strikingly in exosomes within C2 gene set. One of these immune response pathways, MHC-1 antigen presentation, was previously proved to interact with eATP synthase which could be recognized by several immune cells. We used A549-derived exosomes treating human lymphoma Jurkat T cells and found that the proliferation of Jurkat T cells was decreased. Additionally, exosomes isolated from serum-depletion A549-cultured medium strongly declined the proliferation of Jurkat T cells then exosomes from normal A549-cultured medium. To further investigate how eATP synthase in exosomes affect the proliferation of Jurkat T cell, we searched our previous protein array data (Tsai, 2012), and comparing to the Jurkat T cells proteomic data (Wu et al, 2007). We found out FYN, a kinase located at the plasma membrane of Jurkat T cell, which is related to T cell receptor signaling. We suggested that FYN might interact with eATP synthase in exosomes, and affect the proliferation of Jurkat T cells. We used protein-protein docking to predict if they interacted or not, and our results showed that there was a great possibility. We further used anti-ATP synthase antibodies to block the eATP synthase on the exosomes, and the results suggested that eATP synthase on the exosomes related to the proliferation of Jurkat T cells. Whereas, we used the anti-FYN antibodies to block the FYN on the Jurkat T cells, and the results indicated that even if we treated the exosomes isolated from serum deprivation media, the proliferation of Jurkat T cells was not reduced. But the accurate mechanism needed to experiment. In summary, our results suggest that eATP synthase on EVs under serum deprivation may play an important role in the immune response via eATP synthase on exosome surface interacting with FYN on Jurkat T cell surface.

誌謝 i
中文摘要 ii
ABSTRACT iv
CONTENTS vi
List of Figures viii
List of Tables ix
Introduction 1
1.1 Ectopic ATP synthase 1
1.2 Extracellular vesicles 2
1.3 Motivation 3
Materials and Methods 4
2.1 Cell culture 4
2.2 Drug treatment 4
2.3 EV isolation 4
2.4 Transmission Electron Microscopy (TEM) 5
2.5 Nanoparticle Tracking Analysis (NTA) 5
2.6 Western blot 6
2.7 Dot blot 6
2.8 ATP detection assay 7
2.9 Protein precipitation 7
2.10 Protein separation and digestion 7
2.11 Dimethyl labeling of peptides 8
2.12 Desalting with SDB-XC stage tip 8
2.13 SCX Fractionation 9
2.14 NanoLC-MS/MS analysis 9
2.15 Proteome data analysis 10
2.16 Gene set enrichment analysis (GSEA) 11
2.17 Proliferation assay of Jurkat T cells 11
2.18 Protein-protein docking simulation 12
2.19 Statistical analysis 12
Results 14
3.1 Characterization of A549-derived microvesicles and exosomes 14
3.2 Serum deprivation increased expression of eATP synthase on the plasma membrane and EVs 14
3.3 The function of ATP synthase in EVs 15
3.4 Serum deprivation increased immune function in exosomes 16
3.5 A549-derived exosomes reduced the proliferation of human lymphoma Jurkat T cells 17
3.6 eATP synthase on exosome bound to FYN on Jurkat T cell 17
Discussion 20
Conclusion 22
REFERENCE 23
List of Figures
Figure 1. Isolation of each subtypes of extracellular vesicles secreted by A549 lung cancer cells………………………………………………………………….…………..26
Figure 2. Serum deprivation increase eATP synthase and secretion of EVs…………….27
Figure 3. Detection of extracellular ATP produced by eATP synthase of EVs………….28
Figure 4. Proteomic data indicates that serum deprivation alters the content of protein in EVs……………………………………………………………………………………..29
Figure 5. A549-derived exosomes reduced proliferation of Jurkat cells…………….….31
Figure 6. FYN interacts with ATP5B……………..……………………………….……32
Figure 7. Anti-FYN antibodies interfered in A549-derived exosomes interacting with Jurkat T cells……………….………………….………………………………………..34
List of Tables
Table 1. The List of protein identified in microvesicles……………………………….35
Table 2. The List of protein identified in exosomes……………………………………53
Table 3. The List of protein identified in cells…………………………………………79
Table 4. The list of protein related to REACTOME_class I MHC mediated antigen processing presentation…………………………………………………………….… 145
Table 5. The list of protein related to REACTOME_adaptive immune system………147

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