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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孟子青(Tzu-Ching Meng) | |
| dc.contributor.author | Chun-Yi Yang | en |
| dc.contributor.author | 楊君怡 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:59:51Z | - |
| dc.date.copyright | 2018-08-18 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-09 | |
| dc.identifier.citation | 1. Jagannathan, L., S. Cuddapah, and M. Costa, Oxidative stress under ambient and physiological oxygen tension in tissue culture. Curr Pharmacol Rep, 2016. 2(2): p. 64-72.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22050 | - |
| dc.description.abstract | 活性氧化物質在心臟病生理學的發展和進展中扮演關鍵作用。蛋白質是心肌細胞中產生的細胞活性氧化物質的主要標定之一。在20種常見的胺基酸中，半胱胺酸因為具有低解離常數值特性所以非常容易進行氧化反應。基於半胱胺酸座落在蛋白質功能基位點，而這類型蛋白質於病生理中扮演著重要的角色，所以顯示出定義硫醇修飾的氧化還原作用角色的重要性。在本論文裡，我們的研究重心探討的是不可逆半胱胺酸磺酸化會對於蛋白質酪氨酸磷酸水解酶進行氧化還原的調控。蛋白質酪氨酸磷酸水解酶活化位點的半胱胺酸具有低解離常數值特性,因此這類酵素的半胱胺酸也很容易被內生性活性氧化物質直接標定。為了研究在有活性氧化物質誘導下，半胱胺酸磺酸化對於蛋白質酪氨酸磷酸水解酶的影響以及蛋白質品量控管機制下蛋白水解的機制。我們使用了可以直接偵測蛋白質半胱胺酸磺酸化的特定抗體，並且發現在H9c2細胞中內生性蛋白質酪氨酸磷酸水解酶不可逆氧化的存在並且會走向蛋白酶體的降解。蛋白質酪氨酸磷酸水解酶1B（PTP1B）是這類酵素中在半胱胺酸氧化還原研究方面較完整，所以我們選擇PTP1B作為我們的實驗模型。透過細胞內和細胞外免疫沉澱分析方法，我們發現在有H2O2的處理下,PTP1B活化位點Cys215會被磺酸化，而這類型的氧化修飾進而有利於PTP1B泛素化修飾與其蛋白水解。然而我們將PTP1B活化位點Cys215進行性定點突變則會抑制PTP1B被磺酸化進而消除了PTP1B泛素化修飾與其降解。進一步我們透過高通量酵母菌蛋白質體晶片，我們找出與人類蛋白質Cul1 E3連接酶同源的酵母菌蛋白質CDC53可以和具有半胱胺酸磺酸化的化學合成胜肽進行強烈地交互作用。我們隨後在Cul1 E3連接酶的顯性失活結構體實驗顯示出透過Cul1對於PTP1B活化位點Cys215磺酸化的辨認進而將PTP1B視為Cul1的受質。當我們抑制Cul1針對半胱胺酸磺酸化蛋白質酪氨酸磷酸水解酶的蛋白質降解會則增加心臟毒性的產生。這些結果提供了生物學上的半胱胺酸氧化修飾意義，而這種氧化修飾會促使心肌蛋白質經由泛素/蛋白酶體途徑進行蛋白質代谢。 | zh_TW |
| dc.description.abstract | Reactive oxygen species (ROS) has been shown to play a critical role in the development and progression of cardiac pathophysiology. Protein is one of the prime targets of cellular ROS generated in cardiomyocytes. Among the 20 common amino acid, cysteine (Cys) residue, which has a remarkably low pKa characteristic, is highly susceptible to oxidation. Identification of the redox role of thiol modifications has gained significant importance because Cys residue plays an important role in protein function under pathophysiological conditions. Here, our study focuses on the redox-dependent regulation of Cys-sulfonation (SO3H), which belongs irreversible oxidation, on protein tyrosine phosphatases (PTPs). Because of its high nucleophilic property, catalytic Cys residue within PTPs is the direct targets of cellular ROS. To investigate the effect of ROS-induced Cys-sulfonation on PTPs and the mechanism of proteolysis in protein quality control machinery. Using the antibody-based method that could detect protein Cys-sulfonation, we found that endogenous PTPs already oxidized irreversibly and undergoing proteasome-mediated degradation in H9c2 cells. Protein tyrosine phosphatase 1B (PTP1B) is one of PTPs oxidation well-studied, so we chose PTP1B as our experiment model. Upon H2O2 stimuli, through the in vivo and in vitro immunoprecipitation assays, we found that PTP1B catalytic Cys215 residue was sulfonated and it facilitated PTP1B for ubiquitination and proteolysis. Inhibition of PTP1B oxidation by site-directed mutagenesis of PTP1B at active-site Cys215 abrogates the effects of protein ubiquitination and degradation on PTP1B. Through the high-throughput Yeast proteome chips, we identified CDC53, which has homology to human Cul1 E3 ligase, could strongly interact with the chemically synthesized peptide containing Cys-sulfonation. Subsequent investigation with the dominant-negative construct of Cul1 E3 ligase suggested that PTP1B serve as the substrate for Cul1 via Cys-sulfonation recognition. Impairment of Cul1-mediated degradation of Cys-sulfonated PTPs potentiated cardiotoxicity. The findings of this study provide important insight into how biologically significant Cys oxidation directs myocardial protein turnover through ubiquitin/proteasome-mediated degradation. | en |
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| dc.description.tableofcontents | TABLE OF CONTENTS
致謝...................................................................................................................................i 中文摘要........................................................................................................................ii ABSTRACT..................................................................................................................iii ABBREVIATION.......................................................................................................iv TABLE OF CONTENTS............................................................................................v LIST OF FIGURES..................................................................................................viii LIST OF TABLE..........................................................................................................x LIST OF SCHMEMS..................................................................................................x CHAPTER 1: INTRODUCTION.............................................................................1 1.1 Physiological ROS are continuously generated in the heart..................................2 1.2 The Cys-based proteins.............................................................................................5 1.3The PTP superfamily..................................................................................................7 1.4 The character of the active site Cys in PTP............................................................9 1.5 Oxidative modifications of PTPs............................................................................10 1.6 The antibody-based method for monitoring PTP irreversible oxidation...........12 1.7 The protein quality control.....................................................................................13 1.8 Degradation of oxidized proteins...........................................................................16 1.9 Focuses of this study................................................................................................18 CHAPTER 2: MATERIALS AND METHODS................................................20 2.1 Reagents....................................................................................................................21 2.2 Cell culture...............................................................................................................22 2.3 Primary cell culture.................................................................................................22 2.4 Transient transfection.............................................................................................22 2.5 In vivo ubiquitination assay....................................................................................23 2.6 Immunoprecipitation...............................................................................................23 2.7 Immunoblotting and antibodies.............................................................................24 2.8 Immunofluorescence microscopy...........................................................................25 2.9 In vitro-oxidized PTP1B..........................................................................................26 2.10 In vitro ubiquitination assay.................................................................................26 2.11 Fabrication of yeast proteome chip......................................................................27 2.12 Yeast proteome chip assays with biotin-labeled peptides..................................28 2.13 LDH cytotoxicity assay..........................................................................................29 2.14 Statistical analysis..................................................................................................29 CHAPTER 3: RESULTS AND DISCUSSION..................................................31 3.1 Sulfonation of catalytic Cys residue drives PTP degradation.............................32 3.2 PTP1B is targeted for proteasomal degradation..................................................33 3.3 Cys215 residue of PTP1B is targeted for ubiquitin-mediated proteasomal degradation.....................................................................................................................34 3.4 Mutation of Cys215 residue in PTP1B suppresses H2O2-induced degradation.....................................................................................................................36 3.5 H2O2-induced ubiquitination and degradation of PTP1B is Cys215 oxidation-dependent manner........................................................................................38 3.6 Cul1 interacts with PTP1B.....................................................................................39 3.7 E3 ligase Cul1 mediates the degradation and ubiquitination of PTP1B............40 3.8 Knockdown of Cul1 induces cardiotoxicity...........................................................42 3.9 Discussion.................................................................................................................43 CHAPTER 4: SUMMARY......................................................................................50 CHAPTER 5: FIGURES AND TABLES............................................................53 CHAPTER 6: REFERENCES.................................................................................77 LIST OF FIGURES INTRODUCTION: Figure I. Postulated model of the metabolic fate of mitochondria1 H2O2..................3 Figure II. The relationship between myocardial oxygen consumption (MVO2) and myocardial levels of H2O2...............................................................................................4 Figure III. Oxidative modifications of protein cysteine residue..................................6 Figure IV. The human PTPome.....................................................................................9 Figure V. The ubiquitin system....................................................................................14 Figure VI. RING-, HECT-, and RBR-type E3 work by different mechanisms....................................................................................................................16 RESULTS: Figure 1. Tissue distribution of endogenous PTPs oxidation in various tissues in mouse..............................................................................................................................54 Figure 2. Endogenous PTPs are constitutively hyperoxidized in H9c2 cells..................................................................................................................................55 Figure 3. Turnover of endogenous PTPs oxidation in H9c2 cells.............................56 Figure 4. Endogenous PTPs oxidation are targeted for proteasomal degradation.57 Figure 5. Identification of 50 kDa oxPTP protein......................................................58 Figure 6. PTP1B is targeted for proteasomal degradation........................................59 Figure 7. Mutation of cysteine residue in PTP1B attenuates Cys-sulfonation formation........................................................................................................................60 Figure 8. Mutation of cysteine residue delays PTP1B turnover................................61 Figure 9. Cys215 of PTP1B is targeted for proteasomal degradation by ubiquitination.................................................................................................................62 Figure 10. H2O2 promotes PTP1B protein degradation.............................................63 Figure 11. Cys215 mutation renders PTP1B resistant to H2O2-induced degradation.....................................................................................................................64 Figure 12. Cys215 mutation hinders the effects of H2O2-induced PTP1B ubiquitination.................................................................................................................65 Figure 13. Scheme outlining the procedure of in vitro ubiquitination assay............66 Figure 14. Expression and purification of PTP1B1-400 wild type (WT) and C215S (C/S) mutant proteins....................................................................................................67 Figure 15. H2O2-induced ubiquitination and degradation of PTP1B is Cys215 oxidation-dependent manner........................................................................................68 Figure 16. A schematic representation of Cys-sulfonation recognition protein identification using a Yeast proteome chip.................................................................69 Figure 17. Cul1 interacts with PTP1B in cells............................................................70 Figure 18. H2O2 promotes interaction of Cul1 with PTP1B in cells.........................71 Figure 19. E3 ligase Cul1 mediates the degradation and ubiquitination of PTP1B.............................................................................................................................72 Figure 20. siRNA-mediated knockdown of the Cul1 causes the accumulation of hyperoxidized PTPs.......................................................................................................73 Figure 21. Cul1-knockdown cells exhibit increased cardiotoxicity...........................74 Figure 22. Cul1 knockdown induces cardiotoxicity in response to H2O2 stimulation......................................................................................................................75 LIST OF TABLE Table 1. Cys-sulfonation-interacting proteins identified by Yeast proteome chips................................................................................................................................76 LIST OF SCHEMES Scheme 1. Hypothesis: Cys-sulfonation programs PTP degradation in cardiomyocytes...............................................................................................................19 Scheme 2. Summary: The proposed model of the ROS-induced degradation of PTP..................................................................................................................................52 | |
| dc.language.iso | en | |
| dc.subject | 泛素/蛋白?體機制 | zh_TW |
| dc.subject | 心臟 | zh_TW |
| dc.subject | 半胱胺酸修飾 | zh_TW |
| dc.subject | 半胱胺酸磺酸化 | zh_TW |
| dc.subject | 蛋白質酪氨酸磷酸水解? | zh_TW |
| dc.subject | 蛋白質降解 | zh_TW |
| dc.subject | Ubiquitin/proteasome mechanism | en |
| dc.subject | Heart | en |
| dc.subject | Cys modification | en |
| dc.subject | Cys-sulfonation | en |
| dc.subject | Protein tyrosine phosphatase | en |
| dc.subject | Protein degradation | en |
| dc.title | 半胱胺酸磺酸化誘發酪氨酸去磷酸酶以泛素化降解之研究: 氧化壓力下蛋白質品質控管之分子機制 | zh_TW |
| dc.title | Cysteine sulfonation induces ubiquitin-dependent proteolysis of protein tyrosine phosphatases: A molecular basis for protein quality control under oxidative stress | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳健生(Chien-Sheng Chen),張震東(Geen-Dong Chang),王寧(Ling Wang),邱繼輝(Kay-Hooi Khoo) | |
| dc.subject.keyword | 心臟,半胱胺酸修飾,半胱胺酸磺酸化,蛋白質酪氨酸磷酸水解?,蛋白質降解,泛素/蛋白?體機制, | zh_TW |
| dc.subject.keyword | Heart,Cys modification,Cys-sulfonation,Protein tyrosine phosphatase,Protein degradation,Ubiquitin/proteasome mechanism, | en |
| dc.relation.page | 84 | |
| dc.identifier.doi | 10.6342/NTU201802872 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-08-10 | |
| 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 未授權公開取用 | 2.69 MB | Adobe PDF |
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