藉由離氨酸及半胱氨酸標定的二維差異電泳分析人類肺癌細胞中穀胱甘肽還原酶的角色

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研究生：	范喬媛 Fan, Chiao-Yuan
論文名稱：	藉由離氨酸及半胱氨酸標定的二維差異電泳分析人類肺癌細胞中穀胱甘肽還原酶的角色 Proteomic and redox-proteomic study on the role of glutathione reductase in human lung cancer cells
指導教授：	詹鴻霖
口試委員:	周秀專黃三元
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	78
中文關鍵詞：	穀胱甘肽還原酶、蛋白質體學、氧化還原蛋白質體學
外文關鍵詞：	glutathione reductase, proteomic, redox-proteomic
相關次數：	點閱：3 下載：0
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穀胱甘肽還原酶在細胞中扮演著維持氧化還原衡定狀態的角色。然而全面性的研究穀胱甘肽還原酶所調節細胞中的反應包括蛋白質表現量和氧化還原的調節尚未被詳加闡述。在本研究中，我們培養了人類肺腺癌細胞株CL1-0和穀胱甘肽還原酶基因降低表現的衍生細胞株CL1-0ΔGR來研究這兩株細胞株中差異蛋白質表現量和氧化還原的調節。我們使用了離氨酸及半胱氨酸標定的二維差異電泳結合基質輔助雷射脫附游離飛行式質譜儀 (MALDI-TOF MS) 鑑定出具有表現量差異且具有巰基變化的蛋白質。我們鑑定到了在穀胱甘肽還原酶基因降低表現之後有34個蛋白質具有表現量差異、17個蛋白質具有巰基變化。其中有些具有表現量差異的蛋白質參與了氧化還原的調節、鈣離子訊號、細胞骨架的調控和蛋白質摺疊，而有些具有巰基變化的蛋白質參與了氧化還原的調節、蛋白質摺疊和醣解作用。我們甚至使用CL1-0和CL1-0ΔGR細胞株來鑑定在紫外光照射下這兩株細胞的不同反應，我們鑑定到有18個蛋白質在紫外光照射下CL1-0ΔGR 細胞株有明顯的改變但在CL1-0細胞中則沒有明顯改變。這些蛋白質參與了蛋白質摺疊、代謝、蛋白質合成和氧化還原的調節在紫外光照射下且在穀胱甘肽還原酶基因降低表現有明顯的蛋白質表現量改變。總結來說，現階段的研究使用了廣泛性的蛋白質體學方法鑑定肺腺癌細胞株穀胱甘肽還原酶調控的蛋白質表現量和氧化還原的調節，就我們所知，這是第一個利用蛋白質體學和氧化還原蛋白質體學研究在哺乳動物細胞模型中穀胱甘肽還原酶的角色。

Glutathione reductase (GR) plays an important role in the maintenance of correct redox status in cells. However, the global investigations of GR-modulated cellular responses including protein expressions and redox-regulations have yet to be elucidated. In this study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate differential protein expression and redox-regulation of these two cell lines. We used lysine- and cysteine-labeling 2D-DIGE and MALDI-TOF MS analysis to monitor the protein expression and thiol-reactivity changes. Our approaches revealed that 34 identified proteins showed significant changes in protein expression, and 17 proteins showed significant changes in thiol reactivity, in response to GR-depletion. Several proteins that are involved in redox regulation, calcium signaling, cytoskeleton regulation and protein folding showed significant changes in expression, whereas proteins involved in redox-regulation, protein folding and glycolysis displayed changes in thiol reactivity. Furthermore, we also used CL1-0 and CL1-0ΔGR cells to evaluate their differential response to UVB-irradiation, and we identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins that are involved in protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression under UVB-irradiation with GR-depletion. In summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression and redox-modification. To our knowledge, this is the first global proteomic and redox-proteomic analysis to investigate the role of GR in mammalian cell model.

中文摘要    ii
ABSTRACT    iii
誌謝    iv
TABLE OF CONTENTS    v
LIST OF FIGURES AND TABLES    vii
ABBREVIATIONS    ix
Chapter 1 INTRODUCTION    1
1.1    Role of reactive oxygen species (ROS)    1
1.2    Glutathione (GSH) and glutathione reductase (GR)    2
1.3    Redox-modifications    4
1.4    Overview on proteomics    5
1.5    Aims of this study    13
Chapter 2 MATERIALS AND METHODS    14
2.1    Chemicals and reagents    14
2.2    Cell culture    15
2.3    Functional assays    16
2.4    Proteomic strategies    19
2.5    Validation of identified proteins    27
Chapter 3 RESULTS    31
3.1    Glutathione reductase expression in CL1-0 cells and the glutathione reductase knockdown strain, CL1-0ΔGR cells.    31
3.2    Comparison of protein expression levels between CL1-0 and CL1-0ΔGR cells by lysine-labeling 2D-DIGE analysis    34
3.3    Identification of differentially expressed proteins using MALDI-TOF-MS analysis    37
3.4    Validation of differentially expressed proteins identified through expression proteomic study in CL1-0 and CL1-0ΔGR cells    42
3.5    Redox proteomic analysis of GR depletion-induced cysteine modifications in CL1-0 proteins    45
3.6    Identification of thiol reactive proteins using MALDI-TOF-MS analysis    47
3.7    Validation of thiol reactive proteins identified through redox-proteomic study in CL1-0 and CL1-0ΔGR cells    49
3.8    Effect of UVB irradiation on cell viability    50
3.9    Effect of UVB induced intracellular ROS level    51
3.10    Comparison of protein expression levels between CL1-0 and CL1-0ΔGR cells in response to UVB irradiation by lysine-labeling 2D-DIGE analysis    53
3.11    Identification of thiol reactive proteins using MALDI-TOF-MS analysis    55
3.12    Validation of differentially expressed proteins identified through expression proteomic study in UVB-irradiated CL1-0 and CL1-0ΔGR cells    57
Chapter 4 DISCUSSIONS    59
Chapter 5 CONCLUSIONS    66
Chapter 6 REFERENCE    67
APPENDIX    71
Supplementary table 1    72
Supplementary table 2.    75
Supplementary table 3    77

                                

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