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：2 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

                                

1. Ray PD, Huang B-W, Tsuji Y: Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cellular Signalling 2012, 24(5):981-990.
2. Chen K, Keaney JF, Jr.: Evolving concepts of oxidative stress and reactive oxygen species in cardiovascular disease. Curr Atheroscler Rep 2012, 14(5):476-483.
3. Madamanchi NR, Vendrov A, Runge MS: Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005, 25(1):29-38.
4. Vassalle C, Bianchi S, Battaglia D, Landi P, Bianchi F, Carpeggiani C: Elevated levels of oxidative stress as a prognostic predictor of major adverse cardiovascular events in patients with coronary artery disease. J Atheroscler Thromb 2012, 19(8):712-717.
5. Aliev G, Smith MA, de la Torre JC, Perry G: Mitochondria as a primary target for vascular hypoperfusion and oxidative stress in Alzheimer's disease. Mitochondrion 2004, 4(5-6):649-663.
6. Aliev G, Smith MA, Obrenovich ME, de la Torre JC, Perry G: Role of vascular hypoperfusion-induced oxidative stress and mitochondria failure in the pathogenesis of Azheimer disease. Neurotox Res 2003, 5(7):491-504.
7. Bennett S, Grant MM, Aldred S: Oxidative stress in vascular dementia and Alzheimer's disease: a common pathology. J Alzheimers Dis 2009, 17(2):245-257.
8. Gandhi S, Abramov AY: Mechanism of oxidative stress in neurodegeneration. Oxid Med Cell Longev 2012, 2012:428010.
9. Peterson LJ, Flood PM: Oxidative stress and microglial cells in Parkinson's disease. Mediators Inflamm 2012, 2012:401264.
10. Calabrese V, Cornelius C, Leso V, Trovato-Salinaro A, Ventimiglia B, Cavallaro M, Scuto M, Rizza S, Zanoli L, Neri S et al: Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2 diabetes. Biochim Biophys Acta 2012, 1822(5):729-736.
11. Ceriello A, Novials A, Ortega E, La Sala L, Pujadas G, Testa R, Bonfigli AR, Esposito K, Giugliano D: Evidence that hyperglycemia after recovery from hypoglycemia worsens endothelial function and increases oxidative stress and inflammation in healthy control subjects and subjects with type 1 diabetes. Diabetes 2012, 61(11):2993-2997.
12. de MBS, da Fonseca LJ, da SGG, Rabelo LA, Goulart MO, Vasconcelos
67
SM: Oxidative stress as an underlying contributor in the development of chronic complications in diabetes mellitus. Int J Mol Sci 2013, 14(2):3265-3284.
13. Kryston TB, Georgiev AB, Pissis P, Georgakilas AG: Role of oxidative stress and DNA damage in human carcinogenesis. Mutat Res 2011, 711(1-2):193-201.
14. Mates JM, Segura JA, Alonso FJ, Marquez J: Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch Toxicol 2008, 82(5):273-299.
15. Finkel T, Holbrook NJ: Oxidants, oxidative stress and the biology of ageing. Nature 2000, 408(6809):239-247.
16. Bray TM, Taylor CG: Tissue glutathione, nutrition, and oxidative stress. Can J Physiol Pharmacol 1993, 71(9):746-751.
17. Chakravarthi S, Jessop CE, Bulleid NJ: The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep 2006, 7(3):271-275.
18. Kim SJ, Jung HJ, Hyun DH, Park EH, Kim YM, Lim CJ: Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells. Biochimie 2010, 92(8):927-932.
19. Urso ML, Clarkson PM: Oxidative stress, exercise, and antioxidant supplementation. Toxicology 2003, 189(1-2):41-54.
20. Hancock JT: The role of redox mechanisms in cell signalling. Mol Biotechnol 2009, 43(2):162-166.
21. Barford D: The role of cysteine residues as redox-sensitive regulatory switches. Curr Opin Struct Biol 2004, 14(6):679-686.
22. Mieyal JJ, Chock PB: Posttranslational modification of cysteine in redox signaling and oxidative stress: Focus on s-glutathionylation. Antioxid Redox Signal 2012, 16(6):471-475.
23. Wang Y, Yang J, Yi J: Redox sensing by proteins: oxidative modifications on cysteines and the consequent events. Antioxid Redox Signal 2012, 16(7):649-657.
24. O'Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975, 250(10):4007-4021.
25. Gorg A, Drews O, Luck C, Weiland F, Weiss W: 2D with IPGs. Electrophoresis 2009, 30 Suppl 1:S122-132.
26. Chan HL, Gharbi S, Gaffney PR, Cramer R, Waterfield MD, Timms JF: Proteomic analysis of redox- and ErbB2Dpendent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling
68
two-dimensional difference gel electrophoresis. Proteomics 2005, 5(11):2908-2926.
27. Madureira PA, Hill R, Miller VA, Giacomantonio C, Lee PW, Waisman DM: Annexin A2 is a novel cellular redox regulatory protein involved in tumorigenesis. Oncotarget 2011, 2(12):1075-1093.
28. Jarvis RM, Hughes SM, Ledgerwood EC: Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells. Free Radic Biol Med 2012, 53(7):1522-1530.
29. Wadhwa R, Taira K, Kaul SC: An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where? Cell Stress Chaperones 2002, 7(3):309-316.
30. Flachbartova Z, Kovacech B: Mortalin - a multipotent chaperone regulating cellular processes ranging from viral infection to neurodegeneration. Acta Virol 2013, 57(1):3-15.
31. Rohde M, Daugaard M, Jensen MH, Helin K, Nylandsted J, Jaattela M: Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev 2005, 19(5):570-582.
32. Morimoto RI, Sarge KD, Abravaya K: Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses. J Biol Chem 1992, 267(31):21987-21990.
33. Li H, Liu L, Xing D, Chen WR: Inhibition of the JNK/Bim pathway by Hsp70 prevents Bax activation in UV-induced apoptosis. FEBS Lett 2010, 584(22):4672-4678.
34. Kim K, Kim H, Jeong K, Jung MH, Hahn BS, Yoon KS, Jin BK, Jahng GH, Kang I, Ha J et al: Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress. Apoptosis 2012, 17(8):784-796.
35. Kim J, Choi TG, Ding Y, Kim Y, Ha KS, Lee KH, Kang I, Ha J, Kaufman RJ, Lee J et al: Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca2+ homeostasis after ER stress. J Cell Sci 2008, 121(Pt 21):3636-3648.
36. Tsujimoto Y, Shimizu S: The voltage-dependent anion channel: an essential player in apoptosis. Biochimie 2002, 84(2-3):187-193.
37. Alfonso P, Canamero M, Fernandez-Carbonie F, Nunez A, Casal JI: Proteome analysis of membrane fractions in colorectal carcinomas by using 2D-DIGE saturation labeling. J Proteome Res 2008, 7(10):4247-4255.
38. Freund A, Laberge RM, Demaria M, Campisi J: Lamin B1 loss is a
69
senescence-associated biomarker. Mol Biol Cell 2012, 23(11):2066-2075.

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