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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張震東(Geen-Dong Chang) | |
| dc.contributor.author | Yu-Jung Lee | en |
| dc.contributor.author | 李昱蓉 | zh_TW |
| dc.date.accessioned | 2021-05-12T09:33:15Z | - |
| dc.date.available | 2019-08-07 | |
| dc.date.available | 2021-05-12T09:33:15Z | - |
| dc.date.copyright | 2018-08-07 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-01 | |
| dc.identifier.citation | [1] R.J. Mailloux, S.L. McBride, M.E. Harper, Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics, Trends Biochem Sci 38 (2013) 592-602.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/handle/123456789/1143 | - |
| dc.description.abstract | 氧化壓力與許多疾病與生理功能有關，如癌症、心血管疾病、神經退化疾病。當細胞處於氧化壓力下，蛋白質中半胱胺酸(cysteine)殘基之巰基(thiol group)易受攻擊產生氧化相關之轉譯後修飾(oxidative post-translational modification)，進而影響其功能與結構特性。因此針對蛋白質半胱胺酸殘基之氧化修飾，已有許多功能之探討及鑑定工具之開發。
半胱胺酸氧化修飾包括可逆(reversible)修飾如形成雙硫鍵(disulfide)或次磺酸(sulfenation)，在更高的氧化壓力下則可能形成不可逆的亞磺酸和磺酸(sulfination and sulfonation)。亞磺酸還原酶(sulfiredoxin)為目前被報導唯一可對半胱胺酸亞磺酸化進行還原的酵素，其已知作用對象為過氧化物還原酶(peroxiredoxin 1-4)。在這裡我們利用對細胞處理二醯胺 (diamide)，以引發針對巰基攻擊的氧化修飾，進而探討蛋白質半胱胺酸氧化修飾現象。我們在HeLa細胞中對二醯胺引發之氧化攻擊建立基礎了解，包括蛋白質氧化程度改變以及細胞抗氧化反應的啟動。接著我們再進一步運用此方法探討亞磺酸還原酶之抗氧化功能，利用SRXN1敲除(knockout)之HAP-1細胞和過表現SRXN1之HEK細胞兩種模式，我們報導了亞磺酸還原酶對於二醯胺引發之蛋白質氧化修飾具有保護作用，並探討亞磺酸還原酶在抗氧化反應中之角色，以及亞磺酸還原酶是否與其他抗氧化酵素，如硫氧化還原蛋白還原酶(thioredoxin)，有交互作用。 此外，我們針對改對良蛋白質半胱胺酸之氧化修飾之標定方式，以發展氧化程度之定量描述方式。目前對於蛋白質氧化修飾之偵測工具，主要是針對特定修飾的辨識和捕捉。我們優化PEG- maleimide之反應條件，對蛋白質還原態之半胱胺酸殘基進行標記，藉由免疫轉漬(immunoblotting)後積分其訊號以計算氧化的比例，並提出加權的方式將蛋白質的不同氧化程度計分。我們於生物模型驗證此方式，如胰島素引發之蛋白質酪胺酸磷酸酶1B (PTP1B)氧化，惟此方法仍受如抗體辨識區域遮蔽之限制。 | zh_TW |
| dc.description.abstract | Oxidative stress is relevant to several physiological functions and diseases, including cancer, cardiovascular disease, and neural degeneration. The reactive oxygen and nitrogen species (ROS/RNS) such as hydrogen peroxide arise as by-products of metabolism, while excessive ROS/RNS results in protein oxidation and signaling which leads to disease.
Cysteine oxidation is the main post-translational modification associated with redox signaling and oxidative stress. Reversible cysteine oxidations include disulfide and sulfenic acid, which will be further oxidized to irreversible sulfinic and sulfonic acid while facing extreme oxidative stress. Sulfiredoxin is the only reported enzyme that reduces sulfinic acid on hyperoxidized peroxiredoxin 1 - 4. Herein we treated cells with diamide, which caused thiol-specific attacks, to investigate the antioxidant role of sulfiredoxin. Utilizing SRXN1 knockout HAP-1 cells and FLAG-SRXN1 over-expressing HEK cells, we reported a protective role of sulfiredoxin in diamide- induced protein thiolation, and investigated its interactions with other antioxidant systems such as thioredoxin and glutathione. Maleimide-polyethylene glycol (m-PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG-switch. Following PEG-switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. We optimized the assay conditions and exploited the applications of PEG-switch in quantitation of the extent of protein thiol oxidation in cells in response to H2O2 and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m-PEG immunoblot. Some restrictions of the method are also indicated. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-12T09:33:15Z (GMT). No. of bitstreams: 1 ntu-107-D00b46001-1.pdf: 4221519 bytes, checksum: 9281368832d1710c21819b5e44729a8f (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書
謝誌 i 摘要 ii Abstract iv Index vi Figure List viii Table List viii Appendix Index ix Abbreviation List x Chapter I. Investigation of the Antioxidant Role of Sulfiredoxin 1 1. Introduction 1 1.1 Oxidative stresses and antioxidant systems in mammalian cells 1 1.2 Protein cysteine residues as the victims of oxidative attacks 3 1.3 Sulfiredoxin reduces peroxiredoxin sulfinylations 5 1.4 Specific aims and significances 7 2. Materials and methods 9 2.1. Chemicals and antibodies 9 2.2. Cell culture 9 2.3. SRXN1 knockout, genomic DNA extraction and sequencing 10 2.4. SRXN1 over-expression 10 2.5 Cell lysate preparation 11 2.6. Fractionation 11 2.7. SDS-PAGE, Coomassie blue staining, and immnunoblotting 12 2.8. Immunofluorescence 12 2.9. RNA extraction and RT-PCR 13 2.10. Immunoprecipitation 13 2.11. Diamide treatment and resin-assisted capture 14 2.12. Hydrogen peroxide-induced oxidation 15 2.13. MTT assay 15 2.14. Anti-glutathione antiserum preparation 15 2.15. Thioredoxin reductase activity assay 16 2.16. In-gel digestion and protein identification by mass spectrometry 16 2.17. Metabolomics analysis by mass spectrometry 18 3. Results 20 3.1. Diamide induced reversible thiolations and antioxidant responses 20 3.2. Sulfiredoxin knockout increased diamide-induced protein thiolations 22 3.3. Sulfiredoxin knockout affected functions of antioxidant enzymes 23 3.3.1. Glutathione system 24 3.3.2. Peroxiredoxin and thioredoxin system 25 3.4. Diamide treatment induced cytosolic sulfiredoxin dimerization 26 3.5 Hypothetic mechanism of sulfiredoxin under thiol-specific oxidative attacks 28 4. Discussion 29 Chapter II. Development of Quantitative Display of Protein Redox Status 32 1. Introduction 32 1.1. Detection tools of protein cysteine oxidations 32 1.2. PEG-maleimide as a cysteine-specific probe 34 1.3. Specific aims and significances 35 2. Materials and methods 36 2.1. Chemicals and antibodies 36 2.2. Cell culture and lysis condition 36 2.3. PEG-maleimide labeling 37 2.4. SDS-PAGE, immunoblotting and reduced/oxidized ratio calculation 37 2.5. Hydrogen peroxide-induced oxidation 38 2.6. Insulin-induced PTP1B oxidation in HeLa cell 38 3. Results 39 3.1. Protocol of PEG tagging 39 3.2 Optimizing tagging efficiency 39 3.3. m-PEG tagging in other redox-sensitive proteins 41 3.4. Quantitating the redox status of proteins in cells 42 3.4.1. H2O2-induced peroxiredoxin and Hsp27 oxidations in HEK cell 44 3.4.2. Insulin-induced PTP1B oxidation in HeLa cell 45 4. Discussion 47 Chapter III. Conclusions and Perspectives 50 Figures 52 Tables 84 References 89 Appendix 96 | |
| dc.language.iso | en | |
| dc.subject | 定量偵測 | zh_TW |
| dc.subject | 半胱胺酸氧化修飾 | zh_TW |
| dc.subject | 亞磺酸還原? | zh_TW |
| dc.subject | 抗氧化反應 | zh_TW |
| dc.subject | 二醯胺 | zh_TW |
| dc.subject | sulfiredoxin | en |
| dc.subject | quantitative detection | en |
| dc.subject | diamide | en |
| dc.subject | antioxidant response | en |
| dc.subject | Cysteine oxidative modification | en |
| dc.title | 蛋白質半胱胺酸氧化修飾之偵測與亞磺酸還原酶抗氧化角色之研究 | zh_TW |
| dc.title | On Protein Cysteine Oxidation Detection and the Roles of Sulfiredoxin in Antioxidant Responses | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳宏文(Hung-Wen Chen),張茂山(Mau-Sun Chang),張?仁(Ching-Jin Chang),李德彥(Der-Yen Lee) | |
| dc.subject.keyword | 半胱胺酸氧化修飾,亞磺酸還原?,抗氧化反應,二醯胺,定量偵測, | zh_TW |
| dc.subject.keyword | Cysteine oxidative modification,sulfiredoxin,antioxidant response,diamide,quantitative detection, | en |
| dc.relation.page | 116 | |
| dc.identifier.doi | 10.6342/NTU201802212 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-08-01 | |
| 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-107-1.pdf | 4.12 MB | Adobe PDF | 檢視/開啟 |
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