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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊宏 | |
| dc.contributor.author | Bing-Yu Chiang | en |
| dc.contributor.author | 江秉諭 | zh_TW |
| dc.date.accessioned | 2021-06-15T05:45:32Z | - |
| dc.date.available | 2020-08-17 | |
| dc.date.copyright | 2010-08-20 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-19 | |
| dc.identifier.citation | 1. Fang, F. C. (2004) Nat Rev Microbiol 2, 820-832
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47025 | - |
| dc.description.abstract | 有些格蘭氏陰性菌中， 麩氨基硫亞精胺 (glutathionylspermidine, Gsp) 的生成與分解是由一個雙功能性酵素麩氨基硫亞精胺合成酶/水解酶 (glutathionylspermidine synthetase/amidase, GspSA) 所催化。然而，Gsp 在菌體中的生理功能至今仍是不清楚。此外，GspSA 中兩個具有相反活性的區塊如何相互調控目前也是個未知謎團。在本論文中，我們首先提出Gsp 會與大腸桿菌中蛋白質上的某些硫基 (thiol) 形成雙硫鍵。此種新的蛋白質後修飾會隨著外界的氧化壓力的上升而增加。隨後的研究發現，過氧化氫會選擇性的抑制 Gsp水解酶的活性，而不會影響Gsp 合成酶的活性。這選擇性抑制的現象會導致細胞內 Gsp 的累積，並進而造成蛋白質 Gsp thiolation 的上升。利用X 光繞射分析與化學修飾，我們進一步解釋的這個 Gsp amidase 的選擇性抑制是來自於催化胺基酸Cys 59 被氧化成 sulfenic acid。由高解析度X光繞射分析指出GspSA 中有數個特殊的氫鍵來穩定此 sulfenic acid。本研究中提出了一套機制來描述在大腸桿菌中Gsp的如何進行調控，並說明其在氧化還原中所扮演的角色。¬而 Grx-/GspSA- 雙基因突變株顯示出對氧化壓力高度敏感，也說明了 Gsp 在對大腸桿菌在氧化壓力扮演的角色。最後，為了瞭解到底有哪些蛋白質會被修飾，我們藉由被生物素標定的亞精胺與大腸桿菌進行培養。大腸桿菌內的 Gsp 合成酶會合成具有生物素標定的 Gsp 並進一步結合到蛋白質上，藉此親合性純化與質譜分析而得到被 Gsp 標定蛋白質的身分與位置。 | zh_TW |
| dc.description.abstract | Certain bacteria synthesize glutathionylspermidine (Gsp), from glutathione (GSH) and spermidine. E. coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, standing for a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated by H2O2 oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA/glutaredoxin null mutants to H2O2 support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli. Additionally, a platform based on metabolic incorporation of biotinated spermidine was developed to identify Gsp Sthiolated proteins. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T05:45:32Z (GMT). No. of bitstreams: 1 ntu-99-D94b46005-1.pdf: 2448536 bytes, checksum: c527514cd73c8e861093fe605dfda279 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | Content
Acknowledgement.................................I Abstract ......................................II Abbreviations.................................III Content........................................IV 1. Introduction 1.1 The role of glutathione in prokaryotic .....1 1.2 Glutathionylspermidine (Gsp) in E. coli and protozoa ................................................4 1.3 The various states of cysteine modification ................................................11 1.4 Protein S-thiolation .......................13 1.5 The motivation of this thesis ..............15 2. Methods and Materials 2.1 Protein expression and purification ........19 2.2 Reagents and chemicals .....................19 2.3 Enzyme activity assay ......................19 2.4 Analytical ultracentrifugation .............21 2.5 Identification of the cysteine-sulfenic acid by dimedone labeling and subsequent mass spectrometric analysis .......................................22 2.6 Conversion of Gsp-disulfide to GSH by the Gsp amidase and GSH reductase ..............................22 2.7 The viabilities of GspSA and glutaredoxin null mutants after H2O2 treatment ...........................23 2.8 In vitro and in vivo Gsp determination in the presence of H2O2 ........................................23 2.9 Detection of protein Gsp S-thiolation in E. coli ................................................24 2.10 Gsp amidase-catalyzed removal of the Spd moiety from Gsp-thiolated proteins .........................25 2.11 Enrichment and identification of Gsp thiolated proteins .......................................26 3. Results 3.1 Probing the catalytic mechanism of Gsp synthetase by site-direct mutagenesis ........................28 3.2 Selective inactivation of Gsp amidase activity by H2O2 and GSNO .......................................29 3.3 Identification of Cys59-sulfenic acid by x-ray crystallography and chemical Modification–Mass Spectrometry ...................................31 3.4 Rapid accumulation of Gsp in vitro and in vivo in the presence of H2O2 ...............................33 3.5 Conversion of Gsp-disulfide to GSH by the Gsp amidase and GSH reductase couple .......................35 3.6 Discovery of Gsp S-thiolated proteins and their conversion to GSH S-thiolated proteins by the action of Gsp amidase ........................................36 3.7 Sensitivities of different E. coli strains to oxidative stress .........................................38 3.8 Gsp synthetase can accept the N8-derivated spermidine analogue as its substrate. .....................39 3.9 Gsp S-thiolation of OxyR ...................40 3.10 Large-scale identification of Protein Gsp S-thiolation .....................................42 4. Discussion 4.1 A proposed model to demonstrate a functional role of GspSA .......................................45 4.2 The unusual cysteine-sulfenic acid is primarily stabilized by a very short H-bond ...........49 4.3 Dissimilar amidase active sites linked to differential redox regulation ............................52 4.3 Large-scale analysis of protein Gsp S-thiolation by using biotinylated spermidine (Spd-biotin) ...53 5. Future Aspects ............................56 6. Reference ............................58 7. Figures, Table and Legends..................62 | |
| dc.language.iso | en | |
| dc.subject | 胺 | zh_TW |
| dc.subject | 麩氨基硫 | zh_TW |
| dc.subject | 亞精 | zh_TW |
| dc.subject | glutathionylspermidine | en |
| dc.title | 探討麩氨基硫亞精胺合成酶/水解酶之反應機制與其在 大腸桿菌的氧化還原中所扮演的角色 | zh_TW |
| dc.title | E. coli Gsp Synthetase/Amidase: Reaction Mechanisms and Its Role in Redox Regulation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王惠鈞,陳玉如,吳蕙芬,賈景山 | |
| dc.subject.keyword | 麩氨基硫,亞精,胺, | zh_TW |
| dc.subject.keyword | glutathionylspermidine, | en |
| dc.relation.page | 88 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-19 | |
| 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 | |
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