簡易檢索 / 詳目顯示

回結果列表
研究生： 林芳齡
Lin, Fang-Ling
論文名稱： 蛋白標記螢光探針揭示碳酸酐酶的胞外域脫落
Protein Labeling Fluorescent Probe to Study Ectodomain Shedding of Carbonic Anhydrases
指導教授： 陳貴通
Tan, Kui-Thong
口試委員: 林俊成
Lin, Chun-Cheng
詹揚翔
Chan, Yang-Hsiang
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2021
畢業學年度： 109
語文別： 中文
論文頁數： 75
中文關鍵詞： 碳酸酐酶 、膜蛋白 、胞外域脫落 、蛋白標記螢光探針
外文關鍵詞： carbonic anhydrases, membrane protein, ectodomain shedding, protein labeling fluorescent probe
相關次數： 點閱：3  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 胞外域蛋白 (ectodomain) 脫落是一種透過蛋白酶將細胞表面蛋白切除並釋放於外部環境的水解形式，此機制已被發現能調節包括細胞黏附、生長因子之訊號傳遞、發炎反應及細胞的存活等多種細胞行為。現今主要是透過免疫學方法分析分泌於培養液中的胞外域蛋白，然而其應用受限仍大。在本篇論文中，我們以親和性蛋白標記螢光探針作為研究胞外域蛋白的新策略。透過此方法，我們首次揭示了腫瘤缺氧指標之一的碳酸酐酶XII (CAXII) 其胞外域的脫落。相較於傳統的免疫學偵測，這類蛋白標記方法除了能夠測量培養液中的胞外域蛋白外，更能藉由細胞影像追蹤螢光訊號以及定量分析細胞表面剩餘的蛋白，提供了更加詳細的胞外域蛋白脫落以及蛋白通量的數據。我們相信此蛋白標記螢光探針的策略能改善免疫學方法的不足，在胞外域蛋白脫落的研究上成為一重要的工具。

    Ectodomain shedding is a form of limited proteolysis where proteases cleave cell surface proteins resulting in the release of the extracellular domain. Cells use this mechanism to regulate a wide variety of cellular events, including growth factor signaling, cell adhesion, inflammatory responses and cell survival. Typically, immunological methods are employed for the detection of ectodomain secreted to the cultured medium. In this thesis, we described a new strategy by using affinity-based protein labeling fluorescent probe to study ectodomain shedding. With this approach, we have revealed in the first time the ectodomain shedding of cell surface carbonic anhydrase XII (CAXII) which is an important biomarker for tumor hypoxia. Several important shedding parameters were determined, including basal and stimulated shedding rates, protein half-life on the cell surface, and metalloprotease responsible for the ectodomain shedding. As compared with the immunological detection methods, the protein labeling approach not only enables detection of ectodomain released to the culture medium, but also real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, providing a more accurate and detail analysis of the ectodomain shedding as well as protein turnover. We believe that this protein labeling fluorescent probe approach can be a versatile tool to complement classical immunological methods to study ectodomain shedding of membrane proteins.

    摘要 Abstract 謝誌 目錄 第一章、緒論-------------------------------1 1-1 細胞訊息傳遞 (Signal Transduction)-----1 1-2 訊息傳遞的機制-------------------------2 1-2-1 磷酸化 (Phosphorylation)------------2 1-2-2 糖基化 (Glycosylation)--------------4 1-2-3 蛋白水解 (Proteolysis)--------------6 1-3 胞外域蛋白脫落 (Ectodomain Shedding)---8 第二章、文獻回顧---------------------------12 2-1 免疫學偵測法 (Immunological Detection Methods)---12 2-1-1 免疫沉澱法 (Immunoprecipitation)---------------12 2-1-2 西方墨點法 (Western Blotting)------------------14 2-1-3 免疫學方法的缺點-------------------------------16 2-2 化學標記方法-------------------------------------17 2-2-1 放射性同位素標記法 (Radioisotopic Labeling Method)---17 2-2-2 生物素標記法 (Biotin Functionalized Method)----------18 2-2-3 非專一性化學標記方法的缺點----------------------------19 2-3 親和性螢光標記-----------------------------------------20 第三章、碳酸酐酶及其標記探針設計----------------------------22 3-1 碳酸酐酶 (Carbonic Anhydrases, CAs)-------------------22 3-2 螢光探針設計------------------------------------------23 第四章、實驗結果與討論-------------------------------------26 4-1 探針之基本性質探討------------------------------------26 4-1-1 探針1與重組蛋白hCAII之反應性測試---------------------26 4-1-2 探針1與重組蛋白hCAII之反應動力學---------------------27 4-1-3 探針1對重組蛋白hCAII之標記效率-----------------------28 4-1-4 探針1於複雜環境下之選擇性測試------------------------30 4-1-5 探針1之抗酯酶水解測試-------------------------------31 4-1-6 探針1之穩定性測試----------------------------------32 4-1-7 探針1之目標蛋白偵測極限-----------------------------34 4-2 探針1之細胞實驗--------------------------------------34 4-2-1 A549及MCF-7細胞影像之反應性實驗---------------------35 4-2-2 A549及MCF-7細胞裂解液之反應性實驗-------------------36 4-2-3 A549及MCF-7細胞影像之時程實驗 (Time-Course Experiments)---37 4-2-4 A549及MCF-7細胞裂解液之時程實驗---------------------------38 4-2-5 A549及MCF-7細胞裂解液與培養液結果之比較------40 4-2-6 A549及MCF-7細胞胞外域蛋白之活性測試----------41 4-2-7 A549及MCF-7細胞培養液之時程實驗-------------41 4-3 細胞之藥物實驗探討----------------------------42 4-3-1 金屬蛋白酶活化及抑制之藥物測試---------------42 4-3-2 A549及MCF-7細胞在PMA藥物活化下之時程實驗-----46 4-3-3 A549細胞於模擬缺氧環境下之時程實驗-----------49 第五章、總結--------------52 第六章、實驗部分----------53 6-1 實驗藥品與儀器--------53 6-2 蛋白質表達與純化------54 6-2-1 蛋白質表達---------54 6-2-2 蛋白質純化---------55 6-3 SDS-PAGE膠體電泳-----56 6-3-1 製膠配方-----------56 6-3-2 膠體電泳過程-------57 6-4 細胞培養及繼代流程----59 6-4-1 培養液與試劑-------59 6-4-2 細胞繼代培養-------59 6-5 細胞裂解液之SDS-PAGE實驗---------60 6-6 培養液之SDS-PAGE電泳分析實驗-----61 6-7 細胞影像實驗--------------------61 6-8 西方墨點法----------------------62 6-9 細胞存活率分析 (MTT試驗)--------63 第七章、參考資料--------------------65 附錄----------------72

    1. Krauss, G., Basics of Cell Signaling. In Biochemistry of Signal Transduction and Regulation., 5th Edition ed.; WILEY-VCH Verlag GmbH & Co.: 2014; pp 978-3.
    2. Heldin, C.-H., Signal Transduction: Multiple Pathways, Multiple Options for Therapy. Stem cells 2001, 19 (4), 295-303.
    3. van der Geer, P., Signal Transduction. In Brenner's Encyclopedia of Genetics (Second Edition), Maloy, S.; Hughes, K., Eds. Academic Press: San Diego, 2013; pp 436-439.
    4. Berridge, M. J.; Irvine, R. F., Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 1984, 312 (5992), 315-321.
    5. Kodis, E. J., et al., First Messengers. eLS: 2012.
    6. Clark, E.; Brugge, J., Integrins and signal transduction pathways: the road taken. Science 1995, 268 (5208), 233-239.
    7. Yaneeporn Signal Transduction Pathways Model. https://commons.wikimedia.org/wiki/File:1Signal_Transduction_Pathways_Model.svg.
    8. Alberts, B., Molecular biology of the cell. Garland science: 2008.
    9. Thingholm, T. E.; Rönnstrand, L.; Rosenberg, P. A., Why and how to investigate the role of protein phosphorylation in ZIP and ZnT zinc transporter activity and regulation. Cell. Mol. Life Sci. 2020, 77 (16), 3085-3102.
    10. Fukami, Y.; Lipmann, F., Reversal of Rous sarcoma-specific immunoglobulin phosphorylation on tyrosine (ADP as phosphate acceptor) catalyzed by the src gene kinase. Proc. Natl. Acad. Sci. U.S.A. 1983, 80 (7), 1872-1876.
    11. Kole, H.; Abdel-Ghany, M.; Racker, E., Specific dephosphorylation of phosphoproteins by protein-serine and-tyrosine kinases. Proc. Natl. Acad. Sci. U.S.A. 1988, 85 (16), 5849-5853.
    12. Graves, J. D.; Krebs, E. G., Protein phosphorylation and signal transduction. Pharmacol. Ther. 1999, 82 (2-3), 111-121.
    13. McCance, K. L.; Huether, S. E., Pathophysiology-E-book: the biologic basis for disease in adults and children. Elsevier Health Sciences: 2018.
    14. Ardito, F.; Giuliani, M.; Perrone, D.; Troiano, G.; Lo Muzio, L., The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review). Int. J. Mol. Med. 2017, 40 (2), 271-280.
    15. Cohen, P., The regulation of protein function by multisite phosphorylation–a 25 year update. Trends Biochem. Sci. 2000, 25 (12), 596-601.
    16. Heinrich, R.; Neel, B. G.; Rapoport, T. A., Mathematical models of protein kinase signal transduction. Mol. Cell 2002, 9 (5), 957-970.
    17. Liebl, M. C.; Hofmann, T. G., Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road. BioEssays 2019, 41 (12), 1900127.
    18. Ohtsubo, K.; Marth, J. D., Glycosylation in cellular mechanisms of health and disease. Cell 2006, 126 (5), 855-67.
    19. Lowe, J. B.; Marth, J. D., A Genetic Approach to Mammalian Glycan Function. Annu. Rev. Biochem. 2003, 72 (1), 643-691.
    20. Lawson, C. A.; Martin, D. R., Animal models of GM2 gangliosidosis: utility and limitations. Appl. Clin. Genet. 2016, 9, 111.
    21. Donohue Jr, T. M.; Osna, N. A., Intracellular proteolytic systems in alcohol-induced tissue injury. Alcohol Res Health 2003, 27 (4), 317.
    22. Ye, Y.; Fortini, M. E. In Proteolysis and developmental signal transduction, Semin. Cell Dev. Biol., Elsevier: 2000; pp 211-221.
    23. Jenal, U.; Hengge-Aronis, R., Regulation by proteolysis in bacterial cells. Curr. Opin. Microbiol. 2003, 6 (2), 163-172.
    24. Malik, I. T.; Brötz-Oesterhelt, H., Conformational control of the bacterial Clp protease by natural product antibiotics. Nat. Prod. Rep. 2017, 34 (7), 815-831.
    25. Ehrmann, M.; Clausen, T., Proteolysis as a Regulatory Mechanism. Annu. Rev. Genet. 2004, 38 (1), 709-724.
    26. Brown, M. S.; Ye, J.; Rawson, R. B.; Goldstein, J. L., Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 2000, 100 (4), 391-398.
    27. Jorissen, E.; De Strooper, B., γ-Secretase and the intramembrane proteolysis of Notch. Curr. Top. Dev. Biol. 2010, 92, 201-230.
    28. Lichtenthaler, S. F.; Lemberg, M. K.; Fluhrer, R., Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments. EMBO J. 2018, 37 (15), e99456.
    29. Müller, S. A.; Scilabra, S. D.; Lichtenthaler, S. F., Proteomic Substrate Identification for Membrane Proteases in the Brain. Front. Mol. Neurosci. 2016, 9, 96.
    30. Clark, P., Protease-mediated ectodomain shedding. Thorax 2014, 69 (7), 682-684.
    31. Chow, F. L.; Fernandez‐Patron, C., Many membrane proteins undergo ectodomain shedding by proteolytic cleavage. Does one sheddase do the job on all of these proteins? IUBMB Life 2007, 59 (1), 44-47.
    32. Ehlers, M. R.; Riordan, J. F., Membrane proteins with soluble counterparts: role of proteolysis in the release of transmembrane proteins. Biochemistry 1991, 30 (42), 10065-10074.
    33. Reiss, K.; Saftig, P., The “A Disintegrin And Metalloprotease” (ADAM) family of sheddases: Physiological and cellular functions. Semin. Cell Dev. Biol. 2009, 20 (2), 126-137.
    34. Bartsch, J. W., et al., Tumor necrosis factor-α (TNF-α) regulates shedding of TNF-α receptor 1 by the metalloprotease-disintegrin ADAM8: evidence for a protease-regulated feedback loop in neuroprotection. J. Neurosci. 2010, 30 (36), 12210-12218.
    35. Monaco, C.; Nanchahal, J.; Taylor, P.; Feldmann, M., Anti-TNF therapy: past, present and future. Int. Immunol. 2015, 27 (1), 55-62.
    36. Benjannet, S., et al., Post-translational processing of β-secretase (β-amyloid-converting enzyme) and its ectodomain shedding: the pro-and transmembrane/cytosolic domains affect its cellular activity and amyloid-β production. J. Biol. Chem. 2001, 276 (14), 10879-10887.
    37. Ristori, E.; Donnini, S.; Ziche, M., New Insights Into Blood-Brain Barrier Maintenance: The Homeostatic Role of β-Amyloid Precursor Protein in Cerebral Vasculature. Front. Physiol. 2020, 11, 1056.
    38. Hardy, J.; Allsop, D., Amyloid deposition as the central event in the aetiology of Alzheimer's disease. Trends Pharmacol. Sci. 1991, 12, 383-388.
    39. Migliorini, P., Immunoprecipitation. In Antibody Usage in the Lab, Caponi, L.; Migliorini, P., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 1999; pp 47-60.
    40. Lin, J.-S.; Lai, E.-M., Protein–Protein Interactions: Co-Immunoprecipitation. In Bacterial Protein Secretion Systems: Methods and Protocols, Journet, L.; Cascales, E., Eds. Springer New York: New York, NY, 2017; pp 211-219.
    41. Immunoprecipitation (IP). https://rockland-inc.com/ip-method.aspx.
    42. Holmström, P.; Syrjänen, S.; Laine, P.; Valle, S. L.; Suni, J., HIV antibodies in whole saliva detected by ELISA and Western blot assays. J. Med. Virol. 1990, 30 (4), 245-248.
    43. Hill, A. F., et al., The same prion strain causes vCJD and BSE. Nature 1997, 389 (6650), 448-450.
    44. Tian, X., et al., Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection. J. Virol. 2010, 84 (7), 3373-3381.
    45. Kurien, B. T.; Scofield, R. H., Western blotting. Methods 2006, 38 (4), 283-293.
    46. Wang, Y.; Sul, H. S., Ectodomain Shedding of Preadipocyte Factor 1 (Pref-1) by Tumor Necrosis Factor Alpha Converting Enzyme (TACE) and Inhibition of Adipocyte Differentiation. Mol. Cell. Biol. 2006, 26 (14), 5421-5435.
    47. Rennie, M. J., An introduction to the use of tracers in nutrition and metabolism. Proc Nutr Soc 1999, 58 (4), 935-944.
    48. Sanderson, M. P., et al., ADAM10 Mediates Ectodomain Shedding of the Betacellulin Precursor Activated by p-Aminophenylmercuric Acetate and Extracellular Calcium Influx*. J. Biol. Chem. 2005, 280 (3), 1826-1837.
    49. Jia, L.-J.; Krüger, T.; Blango, M. G.; Kniemeyer, O.; Brakhage, A. A., Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of Aspergillus fumigatus Infection. mSphere 2020, 5 (4), e00535-20.
    50. Kunishima, M., et al., Convenient modular method for affinity labeling (MoAL method) based on a catalytic amidation. Chem. Commun. 2009, (37), 5597-5599.
    51. Penders-van Elk, N. J. M. C.; Versteeg, G. F., 10 - Enzyme-enhanced CO2 absorption. In Absorption-Based Post-combustion Capture of Carbon Dioxide, Feron, P. H. M., Ed. Woodhead Publishing: 2016; pp 225-258.
    52. Berg, J. M.; Stryer, L.; Tymoczko, J. L., Stryer Biochemie. Springer-Verlag: 2015.
    53. Fu, Y., et al., Ultra-thin enzymatic liquid membrane for CO 2 separation and capture. Nat. Commun. 2018, 9 (1), 1-12.
    54. Chiche, J., et al., Hypoxia-Inducible Carbonic Anhydrase IX and XII Promote Tumor Cell Growth by Counteracting Acidosis through the Regulation of the Intracellular pH. Cancer Res. 2009, 69 (1), 358-368.
    55. Mboge, M. Y.; Mahon, B. P.; McKenna, R.; Frost, S. C., Carbonic Anhydrases: Role in pH Control and Cancer. Metabolites 2018, 8 (1), 19.
    56. Chiche, J., et al., Hypoxia-Inducible Carbonic Anhydrase IX and XII Promote Tumor Cell Growth by Counteracting Acidosis through the Regulation of the Intracellular pH. Cancer Res. 2009, 69 (1), 358-368.
    57. Kajanova, I., et al., Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells. Br. J. Cancer 2020, 122 (11), 1590-1603.
    58. Zatovicova, M., et al., Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17. Br. J. Cancer 2005, 93 (11), 1267-76.
    59. Kobayashi, M., et al., CAXII Is a sero-diagnostic marker for lung cancer. PLoS One 2012, 7 (3), e33952.
    60. Güttler, A., et al., Cellular and radiobiological effects of carbonic anhydrase IX in human breast cancer cells. Oncol. Rep. 2019, 41 (4), 2585-2594.
    61. Bleibaum, F., et al., ADAM10 sheddase activation is controlled by cell membrane asymmetry. J. Mol. Cell Biol 2019, 11 (11), 979-993.
    62. Herzog, C.; Haun, R. S.; Ludwig, A.; Shah, S. V.; Kaushal, G. P., ADAM10 is the major sheddase responsible for the release of membrane-associated meprin A. J. Biol. Chem. 2014, 289 (19), 13308-22.
    63. Moss, M. L.; Minond, D., Recent Advances in ADAM17 Research: A Promising Target for Cancer and Inflammation. Mediators Inflamm. 2017, 2017, 9673537.
    64. Li, S., et al., Copper depletion inhibits CoCl 2-induced aggressive phenotype of MCF-7 cells via downregulation of HIF-1 and inhibition of Snail/Twist-mediated epithelial-mesenchymal transition. Sci. Rep 2015, 5 (1), 1-17

    QR CODE