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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊宏 | |
| dc.contributor.author | Fu-Tan Hsieh | en |
| dc.contributor.author | 謝馥檀 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:13:32Z | - |
| dc.date.copyright | 2010-08-19 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-16 | |
| dc.identifier.citation | 1. Quastel, J. H., Stewart, C. P., and Tunnicliffe, H. E. (1923) On Glutathione. IV. Constitution, Biochem J 17, 586-592.
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(2003) 20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated, J Biol Chem 278, 679-685. 31. Silva, G. M., Netto, L. E., Discola, K. F., Piassa-Filho, G. M., Pimenta, D. C., Barcena, J. A., and Demasi, M. (2008) Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome, FEBS J 275, 2942-2955. 32. Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Mengozzi, M., Duffieux, F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., and Ghezzi, P. (2002) Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes, Proc Natl Acad Sci USA 99, 3505-3510. 33. Sullivan, D. M., Wehr, N. B., Fergusson, M. M., Levine, R. L., and Finkel, T. (2000) Identification of oxidant-sensitive proteins: TNF-alpha induces protein glutathiolation, Biochemistry 39, 11121-11128. 34. Brennan, J. P., Miller, J. I., Fuller, W., Wait, R., Begum, S., Dunn, M. J., and Eaton, P. (2006) The utility of N,N-biotinyl glutathione disulfide in the study of protein S-glutathiolation, Mol Cell Proteomics 5, 215-225. 35. Craghill, J., Cronshaw, A. D., and Harding, J. J. (2004) The identification of a reaction site of glutathione mixed-disulphide formation on gammaS-crystallin in human lens, Biochem J 379, 595-600. 36. West, M. B., Hill, B. G., Xuan, Y. T., and Bhatnagar, A. (2006) Protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification, FASEB J 20, 1715-1717. 37. Newman, S. F., Sultana, R., Perluigi, M., Coccia, R., Cai, J., Pierce, W. M., Klein, J. B., Turner, D. M., and Butterfield, D. A. (2007) An increase in S-glutathionylated proteins in the Alzheimer's disease inferior parietal lobule, a proteomics approach, J Neurosci Res 85, 1506-1514. 38. Gao, X. H., Bedhomme, M., Veyel, D., Zaffagnini, M., and Lemaire, S. D. (2009) Methods for analysis of protein glutathionylation and their application to photosynthetic organisms, Mol Plant 2, 218-235. 39. Rouhier, N., Lemaire, S. D., and Jacquot, J. P. (2008) The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation, Annu Rev Plant Biol 59, 143-166. 40. Gravina, S. A., and Mieyal, J. J. (1993) Thioltransferase is a specific glutathionyl mixed disulfide oxidoreductase, Biochemistry 32, 3368-3376. 41. Nordstrand, K., slund, F., Holmgren, A., Otting, G., and Berndt, K. D. (1999) NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism, J Mol Biol 286, 541-552. 42. Lind, C., Gerdes, R., Hamnell, Y., Schuppe-Koistinen, I., von Lowenhielm, H. B., Holmgren, A., and Cotgreave, I. A. (2002) Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis, Arch Biochem Biophys 406, 229-240. 43. Cheng, G., Ikeda, Y., Iuchi, Y., and Fujii, J. (2005) Detection of S-glutathionylated proteins by glutathione S-transferase overlay, Arch Biochem Biophys 435, 42-49. 44. Niture, S. K., Velu, C. S., Bailey, N. I., and Srivenugopal, K. S. (2005) S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography, Arch Biochem Biophys 444, 174-184. 45. Klatt, P., Pineda Molina, E., Perez-Sala, D., and Lamas, S. (2000) Novel application of S-nitrosoglutathione-Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation, Biochem J 349, 567-578. 46. Dubin, D. T. (1959) Evidence for Conjugates between Polyamines and Glutathione in E-Coli, Biochem Bioph Res Commun 1, 262-265. 47. Tabor, H., and Tabor, C. W. (1975) Isolation, characterization, and turnover of glutathionylspermidine from Escherichia coli, J Biol Chem 250, 2648-2654. 48. Bollinger, J. M., Jr., Kwon, D. S., Huisman, G. W., Kolter, R., and Walsh, C. T. (1995) Glutathionylspermidine metabolism in Escherichia coli. Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase, J Biol Chem 270, 14031-14041. 49. Kwon, D. S., Lin, C. H., Chen, S., Coward, J. K., Walsh, C. T., and Bollinger, J. M., Jr. (1997) Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains, J Biol Chem 272, 2429-2436. 50. Pai, C. H., Chiang, B. Y., Ko, T. P., Chou, C. C., Chong, C. M., Yen, F. J., Chen, S., Coward, J. K., Wang, A. H., and Lin, C. H. (2006) Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase, EMBO J 25, 5970-5982. 51. Chiang, B. Y., Chen, T. C., Pai, C. H., Chou, C. C., Chen, H. H., Ko, T. P., Hsu, W. H., Chang, C. Y., Wu, W. F., Wang, A. H., and Lin, C. H. (2010) Protein S-thiolation by glutathionylspermidine (GSP): the role of Escherichia coli gsp synthetase/amidase in redox regulation, J Biol Chem 285, 25345-25353. 52. Barrett, W. C., DeGnore, J. P., Konig, S., Fales, H. M., Keng, Y. F., Zhang, Z. Y., Yim, M. B., and Chock, P. B. (1999) Regulation of PTP1B via glutathionylation of the active site cysteine 215, Biochemistry 38, 6699-6705. 53. Shim, H., and Fairlamb, A. H. (1988) Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata, J Gen Microbiol 134, 807-817. 54. Kosower, N. S., and Kosower, E. M. (1995) Diamide: an oxidant probe for thiols, Methods Enzymol 251, 123-133. 55. Seo, Y. H., and Carroll, K. S. (2009) Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies, Proc Natl Acad Sci USA 106, 16163-16168. 56. Ariyanayagam, M. R., and Fairlamb, A. H. (2001) Ovothiol and trypanothione as antioxidants in trypanosomatids, Mol Biochem Parasitol 115, 189-198. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22183 | - |
| dc.description.abstract | 麩胱甘肽化修飾為一種可逆的蛋白質轉譯後修飾，是指麩胱甘肽上的硫醇與蛋白質半胱胺酸的硫醇間以雙硫鍵連接，藉此避免蛋白質上硫醇進行不可逆的氧化反應，此外也能調節蛋白質活性。但現有研究麩胱甘肽化修飾蛋白質之方法仍有缺點與限制。
大腸桿菌中的麩胱甘肽-精胺質合成酶能催化麩胱甘肽與精胺質間醯胺鍵的形成，產生麩胱甘肽-精胺質。本研究利用轉染技術使哺乳類細胞表現麩胱甘肽-精胺質合成酶，並配合 Spd-biotin 探針偵測哺乳類細胞中被麩胱甘肽修飾的蛋白質。透過免疫螢光染色以及高效液相層析儀分析，可在被轉染麩胱甘肽-精胺質合成酶基因的 293T 細胞中觀察到麩胱甘肽-精胺質合成酶蛋白質的表現及麩胱甘肽-精胺質的產生；除此之外，藉由西方墨點法可以確認：Spd-biotin 可進入被轉染麩胱甘肽-精胺質合成酶的 293T 細胞中與內生性的麩胱甘肽連結形成 Gsp-biotin，進而對蛋白質的硫醇基修飾，由實驗結果推論 Gsp-biotin 作用與麩胱甘肽相似能與蛋白質硫醇以雙硫鍵相連。 我們進一步建立有效率的方法鑑定被麩胱甘肽化修飾的蛋白質；前述帶有 Gsp-biotin 修飾的蛋白質，利用胰蛋白酶作用及 streptavidin-膠體進行專一性純化，再以麩胱甘肽-精胺質水解酶將帶有麩胱甘肽的胜肽由膠體上沖堤下來，最後由質譜儀分析，能得到被麩胱甘肽修飾的蛋白質與修飾位置。我們最終的目標為提供一個效率且有用的方法能大規模分析與鑑定麩胱甘肽化之蛋白質。 | zh_TW |
| dc.description.abstract | Protein S-glutathionylation is the formation of mixed disulfide bonds between the thiols of glutathione (GSH) and protein cysteine residues, representing a post-translational modification of proteins. This special modification protects protein thiols from irreversible oxidation and regulates protein functions. To identify S-glutathionylated proteins is the prerequisite to understand the physiolocical function, but the current progress is mainly restricted by currently available methods.
Glutathionylspermidine synthetase (GspS) catalyzes the amide bond formation between GSH and spermidine to synthesize glutathionylspermidine (Gsp). Using GspS and synthesized spermidine-biotin (Spd-biotin), we developed a new chemo-enzymatic method for probing protein glutathionylation in mammalian cells. Immunoblotting and HPLC analysis indicated that GspS were expressed and functional to produce Gsp in GspS-transfected 293T cells. Spd-biotin was shown to go inside the GspS-transfected 293T cells and subsequently conjugate with endogenous GSH to generate Gsp-biotin. Gsp-biotin S-thiolated proteins were also detected by immunoblotting, suggesting that Gsp-biotin presumably acts like GSH to form mixed disulfide bonds with protein thiols, and that our method is able to effectively label GSH S-thiolated proteins. Meanwhile, we established an efficient procedure to identify GSH S-thiolated proteins. Gsp-biotin S-thiolated proteins were subjected to trypsin digestion, enriched by avidin-based affinity chromatography, hydrolyzed by Gsp amidase to give GSH S-thiolated peptides. Futher liquid chromatography-tandem mass spectrometry analysis led to identification of GSH S-thiolated proteins. Our ultimate goal is to provide an efficient and useful platform to characterize protein S-glutathionylation for large-scale analysis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:13:32Z (GMT). No. of bitstreams: 1 ntu-99-R97b46013-1.pdf: 2497257 bytes, checksum: 0aab68ed273e3c72439826d146117c05 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 縮寫表 i
中文摘要 ii Abstract iii 第一章、緒論 1.1 簡介 Glutathione 1 1.2 簡介 Glutathionylation 2 1.2.1 Glutathionylation 的形成與機制 2 1.2.2 蛋白質 glutathionylation 參與細胞生理與其重要性 5 1.3 現有研究 Glutathionylation 的方法與優缺點 6 1.3.1 [35S]cysteine 同位素標定法 6 1.3.2 Biotinylated GSH 6 1.3.3 Anti-GSH 抗體 7 1.3.4 藉由 Glutaredoxin (Grx) 還原 7 1.3.5 Glutathione S-transferase (GST) 偵測法 8 1.3.6 GSH 或 GSH analogs 固定偵測法 8 1.3.7 總結現有方法之限制 9 1.4 Glutathionylspermidine synthetase/amidase (GspSA) 的簡介 9 1.5 研究策略 11 1.6 實驗流程圖 14 第二章、材料與方法 2.1 實驗藥品 15 2.2 酵素動力學測量 15 2.3 分析 GSNO 與 GspNO 對 PTP1B 修飾位置 17 2.4 GspS-pCMV2B 質體之建立 18 2.5 細胞培養與轉染 (transfection) 18 2.6 高效液相層析 (High performance liquid chromatography) 觀察細胞中 Gsp累積 19 2.6.1 細胞中 thiol 小分子之 monobromobimane (mBBr) 衍生化 19 2.6.2 離子對高效液相層析 (ion-pair HPLC) 20 2.7 [14C]spermidine 標定細胞中 Gsp 修飾蛋白質 21 2.8 細胞生長測試 (cell proliferation assay) 21 2.9 in vivo 標定被 glutathionylation 的蛋白質 22 2.9.1 in vivo 標定 22 2.9.2 蛋白質膠體電泳分析 (Polyacrylamide Gel Electrophoresis) 23 2.9.3 西方墨點法 (Western blotting) 25 2.10 蛋白質體（proteomics）分析 25 第三章、實驗結果與討論 Part I in vitro實驗 3.1 Glutathionylspermidine synthetase (GspS) 對 Spd-biotin 之酵素動力學 27 3.2 GSNO 與 GspNO 二者對 PTP1B 的修飾位置相同 27 Part II in vivo 實驗 3.3 GspS 能夠在 293T 細胞中表現並且具有功能而能觀察到 Gsp 的累積 28 3.4 在轉染 pCMV2B-GspS 的 293T 細胞中能觀察到蛋白質被 Gsp 修飾 28 3.5 在濃度 10 mM 以下 Spd-biotin 對細胞沒有毒害 29 3.6 in vivo標定 glutathionylation 修飾之蛋白質 29 3.6.1 對轉染 pCMV2B-GspS 的 293T 細胞處理 Spd-biotin 能標定到蛋白質 29 3.6.2 給予愈高劑量 Spd-biotin 標定到的訊號強度愈強 30 3.6.3 蛋白質之 Gsp-biotin 標定經還原劑或是 Gsp amidase (GspA) 處理後消失 30 3.6.4 給予氧化壓力會使得標定到的蛋白質增多 31 3.6.5 Grx1 能夠還原部分被 Gsp-biotin 修飾的蛋白質 31 Part III 蛋白質體分析 3.7 蛋白質體分析 33 第四章、結論 34 第五章、參考文獻 37 第六章、圖表與說明 45 | |
| dc.language.iso | zh-TW | |
| dc.title | 利用酵素與化學探針偵測蛋白質麩胱甘肽化修飾 | zh_TW |
| dc.title | Development of a chemo-enzymatic method for probing protein glutathionylation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 邱繼輝,陳玉如 | |
| dc.subject.keyword | 麩胱甘肽,麩胱甘肽,化修飾,麩胱甘肽,-精胺質合成酶,活性氧化分子,蛋白質體, | zh_TW |
| dc.subject.keyword | glutathione,glutathionylation,glutathionylspermidine synthetase,reactive oxygen species,proteomics, | en |
| dc.relation.page | 59 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-08-17 | |
| 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 | |
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