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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
| dc.contributor.author | Ting-Yu Huang | en |
| dc.contributor.author | 黃鼎育 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:37:17Z | - |
| dc.date.available | 2022-08-04 | |
| dc.date.copyright | 2017-08-04 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-07-31 | |
| dc.identifier.citation | 1 Weber, J. & Senior, A. E. Catalytic mechanism of F1-ATPase. Biochimica et Biophysica Acta 1319, 19-58 (1997).
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Nature Protocols 2, 1896-1906 (2007). 32 Vizcaino, J. A., Csordas, A., del-Toro, N., Dianes, J. A., Griss, J., Lavidas, I., Mayer, G., Perez-Riverol, Y., Reisinger, F., Ternent, T., Xu, Q. W., Wang, R. & Hermjakob, H. 2016 update of the PRIDE database and its related tools. Nucleic acids research 44, D447-456 (2016). 33 Subramanian, A., Tamayo, P., Mootha, V. K., Mukherjee, S., Ebert, B. L., Gillette, M. A., Paulovich, A., Pomeroy, S. L., Golub, T. R., Lander, E. S. & Mesirov, J. P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America 102, 15545-15550 (2005). 34 Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628-644 (2009). 35 Dudek, J., Rehling, P. & van der Laan, M. Mitochondrial protein import: common principles and physiological networks. 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Annals of Medicine 38, 429-438 (2006). 41 Quillen, E. E., Haslam, G. C., Samra, H. S., Amani-Taleshi, D., Knight, J. A., Wyatt, D. E., Bishop, S. C., Colvert, K. K., Richter, M. L. & Kitos, P. A. Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells. The Journal of Biological Chemistry 281, 20728-20737 (2006). 42 Arakaki, N., Nagao, T., Niki, R., Toyofuku, A., Tanaka, H., Kuramoto, Y., Emoto, Y., Shibata, H., Magota, K. & Higuti, T. Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells. Molecular Cancer Research 1, 931-939 (2003). 43 Moser, T. L., Stack, M. S., Asplin, I., Enghild, J. J., Højrup, P., Everitt, L., Hubchak, S., Schnaper, H. W. & Pizzo, S. V. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 96, 2811-2816 (1999). 44 Moser, T. L., Kenan, D. J., Ashley, T. A., Roy, J. A., Goodman, M. D., Misra, U. K., Cheek, D. J. & Pizzo, S. V. Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proceedings of the National Academy of Sciences of the United States of America 98, 6656-6661 (2001). 45 Yamamoto, K., Shimizu, N., Obi, S., Kumagaya, S., Taketani, Y., Kamiya, A. & Ando, J. Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells. American Journal of Physiology. Heart and Circulatory Physiology 293, H1646-1653 (2007). 46 Ashkin, A., Schutze, K., Dziedzic, J. M., Euteneuer, U. & Schliwa, M. Force generation of organelle transport measured in vivo by an infrared laser trap. Nature 348, 346-348 (1990). 47 Koonce, M. P. & Schliwa, M. Bidirectional organelle transport can occur in cell processes that contain single microtubules. The Journal of Cell Biology 100, 322-326 (1985). 48 Stone, M. C., Roegiers, F. & Rolls, M. M. Microtubules have opposite orientation in axons and dendrites of Drosophila neurons. Molecular Biology of the Cell 19, 4122-4129 (2008). 49 Melkov, A., Baskar, R., Alcalay, Y. & Abdu, U. A new mode of mitochondrial transport and polarized sorting regulated by Dynein, Milton and Miro. Development 143, 4203-4213 (2016). 50 Smith, J. A., Slusher, B. S., Wozniak, K. M., Farah, M. H., Smiyun, G., Wilson, L., Feinstein, S. & Jordan, M. A. Structural basis for induction of peripheral neuropathy by microtubule-targeting cancer drugs. Cancer Research 76, 5115-5123 (2016). 51 Li, Z., Okamoto, K., Hayashi, Y. & Sheng, M. The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873-887 (2004). 52 Smirnova, E., Griparic, L., Shurland, D. L. & van der Bliek, A. M. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Molecular Biology of the Cell 12, 2245-2256 (2001). 53 Smirnova, E., Shurland, D. L., Ryazantsev, S. N. & van der Bliek, A. M. A human dynamin-related protein controls the distribution of mitochondria. The Journal of Cell Biology 143, 351-358 (1998). 54 Verstreken, P., Ly, C. V., Venken, K. J., Koh, T. W., Zhou, Y. & Bellen, H. J. Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47, 365-378 (2005). 55 Ishihara, N., Nomura, M., Jofuku, A., Kato, H., Suzuki, S. O., Masuda, K., Otera, H., Nakanishi, Y., Nonaka, I., Goto, Yu-ichi, Taguchi, N., Morinaga, H., Maeda, M., Takayanagi, R., Yokota, S. & Mihara, K. Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice. Nat Cell Biol 11, 958-966 (2009). 56 Sheng, Z.-H. & Cai, Q. Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration. Nat Rev Neurosci 13, 77-93 (2012). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67552 | - |
| dc.description.abstract | 三磷酸腺苷合成酶(ATP synthase)是一種位於粒線體內膜上負責產生三磷酸腺苷給細胞內許多反應途徑使用的酵素。我們先前的研究發現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，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。為了研究三磷酸腺苷合成酶的次單元參與在運輸路徑中的狀況，我們利用了霰彈槍蛋白質體學(shot-gun proteomics)的技術來分析肺癌細胞A549的細胞膜蛋白並且找到了四個三磷酸腺苷合成酶的次單元：ATP5A1、ATP5B、ATP5H和MT-ATP6。根據基因集富集分析(gene set enrichment analysis)的結果，我們推測異位表達三磷酸腺苷合成酶的次單元是以組裝好的複合體形式，透過粒線體在細胞中的運輸進而表現在細胞膜上。另一方面，細胞骨架涉及到許多細胞內的運輸。當蛋白質進行運輸時，粒線體和囊泡會藉由運輸蛋白如驅動蛋白(kinesin)和動力蛋白(dynein)的協助，沿著細胞骨架運輸到其目的地。為了探討細胞骨架在異位表達三磷酸腺苷合成酶運輸中的功能，我們利用乳癌細胞MCF-7和肺癌細胞A549，在加了一種讓細胞骨架去聚合化的藥物諾考達唑 (nocodazole)之後，發現不論是免疫螢光染色或是流式細胞儀，都可以看到異位表達三磷酸腺苷合成酶的表現量下降。此外由於驅動蛋白5B (Kinesin family member 5B, KIF5B)對於粒線體的運輸非常重要，我們利用免疫沉澱將驅動蛋白5B及其連結蛋白質純化下來，進一步以液相層析串聯式質譜儀(Liquid chromatography-tandem mass spectrometry, LC-MS/MS)進行分析。結果發現粒線體外膜上負責調控粒線體分裂的動力激活蛋白(Dynamin-1-like protein, Drp1)會與驅動蛋白5B有所連結。綜合上述的實驗結果，我們推測Drp1-KIF5B複合體所調控之粒線體沿著細胞骨架進行的運送，對異位表達三磷酸腺苷合成酶的運輸扮演著非常重要的角色。 | zh_TW |
| dc.description.abstract | Adenosine triphosphate (ATP) synthases which generate ATP for various cellular processes are located on the inner membrane of mitochondria. Our previous studies showed that ATP synthases exist on plasma membrane of several types of cancer cells, which are defined as ectopic ATP synthases. Repressing the expression of ectopic ATP synthases causes cancer cells to go through programmed cell death. However, the trafficking pathway of the ectopic ATP synthase is still unclear. To investigate which ATP synthase subunits are involved in the trafficking process, we performed shot-gun proteomic analysis to reveal the plasma membrane proteins of A549 lung cancer cells and identified four subunits of ATP synthases: ATP5A1, ATP5B, ATP5H, and MT-ATP6. The results of gene set enrichment analysis implied that ATP synthase subunits were assembled as complex on the inner membrane of mitochondria, and then translocated from mitochondria to cell surface.
Motor proteins such as kinesins and dyneins play critical roles in protein trafficking along with microtubules. Both immunocytochemistry and flow cytometry analysis demonstrated that the expression level of ectopic ATP synthase decreased in MCF-7 breast and A549 lung cancer cells treated with nocodazole, a microtubule polymerization inhibitor. Because kinesin family member 5B (KIF5B) is an important motor proteins involved in the transportation of mitochondria, we further performed the immunoprecipitation of KIF5B followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS) to identify the KIF5B-interacting proteins which mediate mitochondrial trafficking. We identified dynamin-1 like proteins (Drp1), one of the mitochondrial outer membrane component, might associate with KIF5B. Taken together, our findings suggest that Drp1-KIF5B complex mediated mitochondrial trafficking along microtubules may play a critical role in the transport of ectopic ATP synthase. Disrupting this Drp1-KIF5B complex might be a potential therapeutic strategy in cancer. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:37:17Z (GMT). No. of bitstreams: 1 ntu-106-R04B43013-1.pdf: 3643807 bytes, checksum: 65816e13463787feef8c813bf81f4e83 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………………………………i
致謝……………………………………………………………………………………...ii 中文摘要………………………………………………………………………………..iii Abstract…………………………………………………………………………………..v List of Abbreviations...…………………………………………………………………vii Contents………………………………………………………………………………....ix List of Tables...…………………………………………………………………………xii List of Figures…………………………………………………………………………xiii Chapter 1 Introduction……...……………………………………………………………1 1.1 Ectopic ATP synthases……………………………………………………………….1 1.2 Microtubule related trafficking in cytosol………..………………………………….2 1.3 Kinesin family member 5B……………….………………………………………….3 1.4 Motivation…………………..……………………………………………………….4 Chapter 2 Materials and Methods………………………………………………………..5 2.1 Experimental design………………………………………………………………....5 2.2 Cell culture………………………………………………………………………......5 2.3 Plasma membrane proteins extraction……………………………………………….6 2.4 Sample preparation for proteome……………………………………………………7 2.4.1 Acetone precipitation………………………………………………………......7 2.4.2 Reduction, alkylation and digestion…………………………………………...7 2.4.3 Desalting with SDB-XC StageTips……………………………………………8 2.4.4 Dimethyl labeling of peptides………………………………………………….9 2.5 NanoLC-MS/MS analysis……………………………………………………………9 2.6 Proteomic data analysis…………………………………………………………….10 2.7 Gene set enrichment analysis……………………………………………………....11 2.8 Immunocytochemistry……………………………………………………………...11 2.9 Flow cytometry…………………………………………………………………......13 2.10 Immunoprecipitation of KIF5B…..……………………………………………….14 2.11 In gel digestion……………………………………………………………………16 2.12 Western blot……………………………………………………………………….16 Chapter 3 Results………………………………………………….……………………18 3.1 Ectopic ATP synthases are overexpressed on the plasma membrane of cancer cells…………………………………………………………………………………......18 3.2 Mitochondria might be involved in the trafficking of ectopic ATP synthases......….19 3.3 Mitochondrial trafficking related genes were up-regulated in the ectopic ATP synthases highly-expressed cancer cells……………………………………………......20 3.4 Disruption of microtubule inhibited the expression of ectopic ATP synthases in cancer cells…………………………………………………………………………......21 3.5 KIF5B-Drp1 complexes were probably involved in the trafficking of mitochondria……………………………………………………………………………22 Chapter 4 Discussion………..………………………………………………………….24 Chapter 5 Conclusion…...……………………………………………………………...29 References……………………………………………………………………………...30 Tables..………………………………………………………………………………….40 Figures..………………………………………………………………………………...89 Appendix I…………………………………………………………………………….100 Appendix II……………………………………………………………………………101 | |
| dc.language.iso | en | |
| dc.subject | 粒線體的運輸 | zh_TW |
| dc.subject | 蛋白質體分析 | zh_TW |
| dc.subject | 異位表達三磷酸腺?合成? | zh_TW |
| dc.subject | 動力激活蛋白(Drp1) | zh_TW |
| dc.subject | 驅動蛋白5B(KIF5B) | zh_TW |
| dc.subject | 細胞骨架 | zh_TW |
| dc.subject | Drp1 | en |
| dc.subject | proteomic analysis | en |
| dc.subject | the trafficking of mitochondria | en |
| dc.subject | microtubule | en |
| dc.subject | KIF5B | en |
| dc.subject | Ectopic ATP synthases | en |
| dc.title | 細胞骨架在異位表達ATP合成酶運輸中扮演的角色 | zh_TW |
| dc.title | The Role of Microtubules in the Trafficking of Ectopic ATP Synthase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃宣誠,王憶卿,李岳倫,賴品光 | |
| dc.subject.keyword | 異位表達三磷酸腺?合成?,蛋白質體分析,粒線體的運輸,細胞骨架,驅動蛋白5B(KIF5B),動力激活蛋白(Drp1), | zh_TW |
| dc.subject.keyword | Ectopic ATP synthases,proteomic analysis,the trafficking of mitochondria,microtubule,KIF5B,Drp1, | en |
| dc.relation.page | 101 | |
| dc.identifier.doi | 10.6342/NTU201701792 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-07-31 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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