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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 翁啟惠 | |
dc.contributor.author | Hsin-Yung Yen | en |
dc.contributor.author | 嚴欣勇 | zh_TW |
dc.date.accessioned | 2021-06-16T08:08:49Z | - |
dc.date.available | 2016-07-22 | |
dc.date.copyright | 2014-07-22 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-05-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58229 | - |
dc.description.abstract | 表皮生長因子接收器為一高度醣化且具有酪氨酸磷酸脢活性的膜蛋白,其在細胞中扮演相當重要的角色,包括調節細胞生長、存活、移動以及轉移等能力。表皮生長因子接收器的活化主要受到表皮生長因子刺激,藉由與表皮生長因子結合,表皮生長因子接收器進行蛋白結構之改變而促進其形成二聚體,二聚體之表皮生長因子接收器進而活化其磷酸脢活性並促使接收器本身之酪氨酸磷酸化,最終活化下游訊號傳遞路徑而影響細胞功能。我們先前的研究指出,肺癌細胞中表皮生長因子接受器之涎酸醣化對於其形成二聚體和磷酸化,具有抑制的作用。為了進一步探討涎酸醣化調控表皮生長因子接收器活化的機制,我們利用生物化學的實驗方法,針對表皮生長因子接受器之活化,包含其與表皮生長因子之結合、二聚體化以及磷酸化,進行動力學和質譜學之探討。研究發現,表皮生長因子接收器之涎酸醣化具有抑制接收器與表皮生長因子結合的作用並進而減緩接收器的二聚體化和酪氨酸磷酸化。特別的是,對於具有抗藥性突變之表皮生長因子接收器 (EGFR L858R/T790M),涎酸醣化能特別抑制其酪氨酸1173號位置之磷酸化,並在帶有此突變的肺癌細胞株中,涎酸醣化且具有增進藥物毒殺之效果。此外,肺癌細胞表面之涎酸醣化也具有調節表皮生長因子接受器絲氨酸與蘇氨酸之功能,代表涎酸醣化同時影響表皮生長因子和其它磷酸脢之交互作用。本篇研究深入探討細胞外部之涎酸醣化調節細胞內部表皮生長因子接收器磷酸化之機制,並對於以表皮生長因子接收器為標靶之治療,提供新的方向與契機。 | zh_TW |
dc.description.abstract | Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR commences conformational changes to dimerize, which results in kinase activation and auto-phosphorylation, and the activation of downstream signaling. Previously, we have demonstrated the suppression effect of EGFR sialylation on its dimerization and phosphorylation in lung cancer cells. To further investigate the mechanism of sialylation on EGFR activation and how sialylation regulates EGFR phosphorylation, we investigated the impact of sialylation on the kinetics of EGF-binding and EGFR dimerization as well as autophosphorylation. The result indicated that sialylation of EGFR suppressed EGF/EGFR association, and this negative regulatory effect was confirmed by downregulation of tyrosine phosphosites in EGFR. Moreover, sialylation showed significant impact on EGFR serine/threonine phosphorylation in lung cancer cells and affected the interactive dynamics between EGFR and other kinases. Of particular interest is a site-specific suppression of pY1173 by sialylation was observed in an EGFR tyrosine kinase inhibitor (TKI)-resistant mutant (L858R/T790M) and the sensitivity to gefitinib was enhanced by sialylation in the lung cancer cell line with this EGFR mutant. Our studies showed a selective effect on the phosphorylation of a TKI-resistant mutant, and this observation may provide insights into a new therapeutic intervention. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T08:08:49Z (GMT). No. of bitstreams: 1 ntu-103-D97b46007-1.pdf: 3025248 bytes, checksum: 8a494640c48f06bd3c5f8c8590732f24 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書……………………………………………………………………………………………. i
誌謝……………………………………………………………………………….…………………………………… ii 中文摘要…………………………………………………………………………………………………………... iii 英文摘要…………………………………………………………………………………………………………… v CHAPTER I: INTRODUCTION……………………………………………………………………. 1 1.1 Epidermal Growth Factor Receptor Family and Their Ligand Specificity……………………………………………………………………………………………………….... 1 1.2 Physical function of EGFR and its ligand redundancy………………………....... 2 1.3 EGFR Dysregulation in Lung Cancer……………………………………………………….. 3 1.4 Mechanism of EGFR Activation………………………………………………………………… 5 1.5 Activation of EGFR Mutants…………………………….......................................... 7 1.6 EGFR-targeted Therapy for NSCLC……………………………............................. 8 1.7 EGFR mutation and therapeutic efficacy of TKIs……………………………………. 11 1.8 Resistance for EGFR TKIs therapy……………………....................................... 14 1.9 EGFR phosphorylation and signaling…………………...................................... 15 1.10 Effect of glycosylation on function of EGFR............................................. 16 AIM…………………………………………………………………………………………………………………… 18 CHAPTER II: RESULTS………………………………………………………………………………… 19 2.1 Effect of Sialylation on EGFR Phosphorylation and Resistance to Tyrosine Kinase Inhibitor Gefitinib……………………………………………………………..... 19 2.1.1 Preparation of sEGFR and desialylated sEGFR……………………………. 19 2.1.2 Investigation of Kinetics for EGFR Dimerization………………………….. 20 2.1.3 EGF-binding Kinetics of sEGFR…………………………………………………….. 22 2.1.4 Effect of Sialylation on EGFR Autophosphorylation…………………….. 23 2.1.5 Regulation of Sialylation on EGFR Phosphorylation in Lung Cancer Cells……………………………………………………………………………………………. 24 2.1.6 The Effect of Sialylation to Autophosphorylation of EGFR Mutants…………………………………………………………………………………………………….. 27 2.1.7 The Effect of Sialylation on Phosphorylation and Gefitinib Sensitivity in Gefitinib Resistant Lung Cancer Cells……………………………… 28 2.2 The Effect of EGFR Fucosylation on the Activation of EGFR……………….… 30 2.2.1 Establishment of A549 Cells with FUT4/FUT6 Overexpression……. 30 2.2.2 Role of Fucosylation on EGFR Activation……………………………………… 31 2.2.3 Role of Fucosylation in EGF-Mediated Cell Invasion……………………. 33 CHAPTER III: DISUSSION…………………………………………………………………………… 34 PERSPECTIVE……………………………………………………………………………………………..... 40 CHAPTER IV MATERIALS AND METHODS……………………………………………. 42 4.1 Cell Culture………………………………………………………………………………………………... 42 4.2 Plasmid Construction and Stable Lines Establishment……………………………. 43 4.3 Purification of FLAG-tagged sEGFR……………………………………………………….. 44 4.4 EGFR Dimerization Assay…………………………………………………………………………. 45 4.5 Molecular Weight Determination by Multi-angle Laser Light Scattering (MALLS) Measurement………………………………………………………………………………….. 45 4.6 Surface Plasmon Resonance (SPR) …………………………………………………………… 47 4.7 In vitro EGFR Phosphorylation Assay………………………………………………………. 48 4.8 Western Blot……………………………………………………………………………………………….. 49 4.9 Proliferation Assay…………………………………………………………………………………….. 50 4.10 EGF-Mediated Invasion Assay………………………………………………………………… 50 4.11 Immunoprecipitation of EGFR from Lung Cancer Cells by Cetuximab-immobilized Beads………………………………………………………………………… 51 4.12 Tip-based pH/acid Controlled Immobilized Metal Ion Affinity Chromatography……………………………………………………………………………………………… 52 4.13 LC-MS/MS Analysis………………………………………………………………………………… 53 4.14 Data Processing and Protein Identification…………………………………………….. 54 4.15 Quantitative Analysis by IDEAL-Q…………………………………………………………. 54 4.16 SWATH-MS Measurement……………………………………………………………………… 55 CHAPTER V FIGURES………………………………………………………………………………….. 57 CHAPTER VI TABLES………………………………………………………………………………….. 87 CHAPTER VII References………………………………………………………………………………. 90 圖目錄 Fig. 1. Gefitinib- and erlotinib-sensitizing mutations of EGFR in NSCLC……. 57 Fig. 2. Mechanism of EGFR activation……………………………………………………………. 58 Fig. 3. Structure of WT and L858R EGFR kinase domain in complex with gefitinib…………………………………………………………………………………………………………….. 59 Fig. 4. Sialylation analysis of sEGFR………………………………………………………………. 60 Fig. 5. Suppression of sEGFR dimerization by sialylation…………………………….. 62 Fig. 6. Suppression of EGF-binding to sEGFR by sialylation……………………….. 63 Fig. 7. In vitro phosphorylation profiling of EGFR………………………………………… 64 Fig. 8. The quantification of phosphopeptides by sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) …… 66 Fig. 9. Responsiveness of EGFR phosphorylation to EGF in lung cancer cells……………………………………………………………………………………………………………………. 68 Fig. 10. Comprehensive identification of EGFR phosphorylation in lung cancer cells……………………………………………………………………………………..................... 70 Fig. 11. In vitro phosphorylation profiling of EGFR mutants…………................ 72 Fig. 12. The effect of sialylation on EGFR phosphorylation and gefitinib sensitivity in lung cancer cell lines with EGFR mutations…………………………….. 74 Fig. 13. Examination of EGFR protein expression and fucosylation in A549 cells with FUT4/FUT6 overexpression…………………………………………………………….. 77 Fig. 14. EGF-induced EGFR dimerization and tyrosine phosphorylation in A549-Mock, -FUT4, and -FUT6 cells………………………………………………………………. 79 Fig. 15. EGFR fucosylation suppresses the dimerization of EGFR……………….. 81 Fig. 16. EGFR dimerization and phosphorylation in A549 cells with FUT8 overexpression………………………………………………………………………………………………….. 82 Fig. 17. Fucosylation suppress EGFR activation to modulate EGF-mediated invasion in lung cancer cells…………………………………………………………………………….. 83 Fig. 18. Site-specific sialylation and fucosylation analysis of EGFR……………… 84 Fig. 19. SSEA4 expression in lung cancer cell lines………………………………………… 85 表目錄 Table 1. Site-specific representative glycans of purified EGFR extracellular domain …………………………………………………………………………………………………………… 87 Table 2. EGFR phosphopeptides identified by LC-MS/MS Analysis…………. 88 Tabel 3. The summary of lung cancer cell lines……………………………………………. 89 | |
dc.language.iso | en | |
dc.title | 表皮生長因子接收器之涎酸醣化對其功能影響之探討 | zh_TW |
dc.title | Effect of Sialylation on EGFR Function and Resistance to Tyrosine Kinase Inhibitor Gefitinib | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 楊泮池,陳玉如,邱繼輝,蕭宏昇 | |
dc.subject.keyword | 涎酸醣化,表皮生長因子接收器,二聚體化,磷酸化,突變,艾瑞莎, | zh_TW |
dc.subject.keyword | EGFR,sialylation,dimerization,phosphorylation,mutation,gefitinib, | en |
dc.relation.page | 97 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-05-19 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 2.95 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。