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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 孟子青 | |
dc.contributor.author | Yi-Yun Chen | en |
dc.contributor.author | 陳怡昀 | zh_TW |
dc.date.accessioned | 2021-06-15T06:54:15Z | - |
dc.date.available | 2013-02-20 | |
dc.date.copyright | 2011-02-20 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-02-11 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48371 | - |
dc.description.abstract | 酪胺酸磷酸水解酶(Protein tyrosine phosphatases, PTPs)家族都具有一個低解離係數特質的半胱胺酸,可催化水解酪胺酸磷酸化作用,並容易受到氧化或是亞硝基化(S-nitrosylation)影響。然而在分析蛋白亞硝基化中遇到最大的困難在於S-NO鍵結十分不穩定,目前為止並沒有可以在複雜樣品中直接偵測亞硝基化的分析技術,因此本研究目的之一是應用質譜分析技術上所具有的高敏感度、高解析度、以及高精準度的優點,搭配定量分析來鑑定蛋白亞硝基化。首先,利用電泳分離技術與介質輔助游離法(MALDI)搭配質譜儀來確定PTP1B活化中心的半胱胺酸可進行亞硝基化反應。此外,利用亞硝基化離子在質譜儀中裂解產生特定質量差距現象開發新穎的層析質譜分析(LC-MS/MS)技術,快速的挑選含有特定質量差距的亞硝基化胜肽離子與其裂解片段,增快鑑定蛋白亞硝基化的速率。質譜分析的結果中,觀察到PTP1B中共有三個半胱胺酸會在一氧化氮供給者刺激時產生亞硝基化的反應,結合定量分析確定最易受到亞硝基化作用的位置位於活化中心的Cys215,同時也以晶體結構分析驗證此結論。此外發現亞硝基化PTP1B有較少的氧化型態,顯現出亞硝基化可以有效的保護磷酸水解酶避免其受到外界氧化壓力的影響,並以此確認亞硝基化也可發生於細胞中。
儘管已有許多臨床與動物實驗證實在急性心肌缺氧環境中一氧化氮具有保護細胞的作用,然而其分子機制尚未明瞭。在此研究中觀察到當內皮細胞在缺氧環境下會釋放出一氧化氮,有助於心肌細胞減少因缺氧所造成的傷害,為了進一步釐清在缺氧環境中一氧化氮所提供的保護機制。選用大鼠心肌細胞(H9c2 rat ventricular myoblast)系統,實驗結果顯示在缺氧環境下H9c2細胞會喪失細胞骨架完整性與擴大細胞接合間隙,同時觀察到酪胺酸磷酸化程度下降與酪胺酸磷酸水解酶的活化,有趣的是當外加一氧化氮供給者(GSNO)到缺氧的H9c2細胞時不僅可以逆轉缺氧所造成的傷害也維持酪胺酸磷酸化的程度,為了鑑定受影響的蛋白質,在細胞培養中利用胺基酸穩定同位素標定技術(SILAC)與層析質譜分析技術來測量酪胺酸磷酸化的變化,結果顯示在缺氧環境下與細胞骨架相關的蛋白質酪胺酸磷酸化程度下降,然而在GSNO刺激下,其酪胺酸磷酸化程度提升回來。此外為了證實一氧化氮可在缺氧環境中經由亞硝基化抑制酪胺酸磷酸水解酶活性,以Biotin Switch技術來偵測亞硝基化的程度。綜合以上的結果推測一氧化氮可能藉由抑制酪胺酸磷酸水解酶活性來影響細胞骨架蛋白的穩定性。利用抑制劑在缺氧狀態下抑制酪胺酸磷酸水解酶的活性,可減緩因缺氧而造成的傷害,證實在缺氧環境中一氧化氮可透過造成酪胺酸磷酸水解酶亞硝基化來維持酪胺酸磷酸化的程度,藉此維持細胞存活率、形態與正常功能。 | zh_TW |
dc.description.abstract | All members in the protein tyrosine phosphatase (PTP) family of enzymes contain an invariant Cys residue which is absolutely indispensable for catalysis. Due to the unique microenvironment surrounding the active center of PTPs, this Cys residue exhibits an unusually low pKa characteristic, thus rendering it highly susceptible to S-nitrosylation. Nevertheless, the direct evidence and the biological consequences for such NO-induced post-translational modification of PTPs are not available. Three strategies were employed in order to reveal the molecular basis that contributes to NO-mediated inactivation of PTPs. First, a gel-based method in conjunction with MALDI-MS was applied to demonstrate that purified PTP1B could be modified by NO. Second, we developed a novel strategy for the determination of S-nitrosylation sites by LC-ESI-MS/MS analysis incorporating a mass difference-based, data-dependent acquisition function that effectively mapped the Snitrosylated Cys residues. Finally, quantitative MS and crystallography-based structural analysis demonstrated that the primary S-nitrosylation site of PTP1B is the active site Cys215.We showed that S-nitrosylation of Cys215 protects PTP1B from further oxidation both in vitro and in vivo. Although the S-nitrosylation of cellular proteins has been reported as a key mechanism for NO-mediated cytoprotective effects in cardiovascular system against hypoxia injury, the underlying mechanism remains elusive. In this study, we observed that, when co-cultured with endothelia, the hypoxia-induced injury of cardiomyocytes was significantly attenuated. One of the cytoprotectors secreted from endothelia was identified as nitric oxide (NO). We have investigated the underlying mechanism through which NO provides the protective effects during cardiac hypoxic injury. Under hypoxic stress, rat myocardia H9c2 cells underwent a loss of F-actin cytoskeletal integrity and intercellular adherens, concomitant with a drastic decrease of protein phosphotyrosine (pTyr) signaling and activation of endogenous protein tyrosine phosphatases (PTPs). Intriguingly, treatment of a physiological form of NO carrier Snitrosoglutathione (GSNO) not only reversed the hypoxia-induced cytoskeletal changes but also sustained pTyr levels of cellular proteins. The stable isotope labeling in cell culture (SILAC) in conjunction with LC-MS/MS analyses identified a cluster of cytoskeletal regulators whose pTyr levels were dependent upon environmental oxygen, yet could be maintained in hypoxia with GSNO supply. Employing the Biotin Switch Method, we further showed that several endogenous PTPs were S-nitrosylated, therefore inactivated by GSNO treatment in cells under the hypoxic condition. Taken together, our data suggested that NO might regulate actin dynamics via inactivation of PTPs. This hypothesis was further confirmed by the direct application of a general PTP inhibitor to cells under hypoxia. Thus, suppression of PTP activity by NO or specific inhibitors may provide a novel therapeutic opportunity for ischemic heart. In summary, we have demonstrated for the first time that NO could protect cardiomyocytes from hypoxic insults through Snitrosylation of endogenous PTPs, thus sustaining the integrity of pTyr signal networks for proper maintenance of cell survival, morphology and function. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:54:15Z (GMT). No. of bitstreams: 1 ntu-100-D95b46005-1.pdf: 12174191 bytes, checksum: 33845d4e1570fb7781283930db9c3a9b (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 口試委員會審定書.................................................................................................................i
誌謝..............................................……………....……………………….……..................ii 中文摘要……………….……………..……………………………………………….…iii Abstract...............................................................................................................................iv Abbreviation........................................................................................................................v Table of Contents………………………………………………………………….….....vi List of Tables.......................................................................................................................xi List of Schemes....................................................................................................................xi List of Figures.....................................................................................................................xii Chapter 1: Introduction.........................................................................................1 1.1 The nitric oxide (NO).................................................................................................... 2 1.1.1 Synthesis and storage..............................................................................................2 1.1.2 The classical intracellular action of NO..................................................................3 1.2 S-nitrosylation.................................................................................................................5 1.2.1 Modulation of protein functions by S-nitrosylation................................................5 1.2.2 Identification of S-nitrosylation proteins and modification sites............................7 1.3 The importance of protein tyrosine phosphatases……………………….…….....10 1.3.1 Tyrosine phosphorylation......................................................................................10 1.3.2 PTP classification..................................................................................................11 1.3.3 Redox regulation of PTP activity..........................................................................12 1.4 Role of NO, S-nitrosylation and PTPs in heart disease............................................13 1.4.1 Cardiovascular disease..........................................................................................13 1.4.2 The role of NO in protection against ischemic/hypoxic injury…………..........14 1.4.3 The role of PTP in cardioprotection against ischemic/hypoxic injury..................16 1.5 Focuses of this study.....................................................................................................17 Chapter 2: Materials and Methods…………….……………...…………19 2.1 Reagents and antibodies……………………………………………………………20 2.2 Cell Culture, transfection, immunoprecipitation, and immunofluorescence staining.......................................................................................................................21 2.3 Labeling or treatment with different reagents………………...…………………23 2.3.1 Preparation of S-nitrosylated and oxidized PTP1B………………………..........23 2.3.2. Reaction with iodoacetic acid and iodoacetamide………………......…………23 2.3.3 Quantitative MS for determining Cys residue susceptibility to S-nitrosylation...23 2.4 Determination of PTP activity……………………………………………………....24 2.4.1 In-gel phosphatase activity assay.........................................................................24 2.4.2 pNPP phosphatase activity assay.........................................................................24 2.4.3 PTP activity assay by using activity-based probe (BBP).....................................25 2.5 Effect of PTP inhibitor (PVS).....................................................................................25 2.6 Crystallization and structural analysis of PTP1B S-nitrosylation...........................26 2.7 Measurement of intracellular NO level......................................................................26 2.8 The modified biotin switch method............................................................................27 2.9 Identification of S-nitrosylated cysteine residues by MS analysis...........................27 2.9.1 In-gel and in-solution digestion............................................................................27 2.9.2 MS analysis for identifying S-nitrosylated sites...................................................28 2.9.3 Filter aided proteome preparation (FASP) and enrichment of phosphotyrosine containing peptides...............................................................................................29 2.9.4 MS analysis for identifying phosphorylated sites................................................29 2.10 MS data analysis by MaxQuant software................................................................30 2.11 Statistics......................................................................................................................31 Chapter 3: Results and Discussion................................................................31 3.1 Part 1: To develop analytical platform for direct identification of Snitrosylated Cys residues on PTP and confirm the presence of Snitrosylated PTPs in intact cells...............................................................................32 3.1.1 Application of a gel-based method that reveal NO donor-induced reversible inactivation of PTP1B...........................................................................................32 3.1.2 Identification of S-nitrosylated cysteine residues in PTP1B by ESI-MS/MSMS analysis..................................................................................................................33 3.1.3 Quantitative MS and structural analysis identified the catalytic Cys215 of PTP1B as the most susceptible S-nitrosylation site...........................................................36 3.1.4 S-nitrosylation protects the active site against irreversible oxidation...................37 3.1.5 Increased cellular NO levels protect endogenous PTP1B against permanent inactivation by oxidation.......................................................................................39 3.1.6 Discussion.............................................................................................................41 3.1.7 Tables, schemes and figures..................................................................................45 3.2 Part II: To investigate S-nitrosylation of PTPs in regulating signaling homeostasis of cardiomyoblasts under hypoxic condition.......................60 3.2.1 Hypoxia initiates morphological change in H9c2 cardiomyoblast cells...............60 3.2.2 Endogenous PTPs in H9c2 cardiomyoblast cells under hypoxia are more active compared to the normoxic cells............................................................................61 3.2.3 Nitric oxide released from MS-1 endothelial cells may decrease hypoxia-induced injury in H9c2 cardiomyoblast cells......................................................................62 3.2.4 Effects of exogenous NO donor on cytoskeleton organization, pTyr levels, and PTP activity under hypoxia condition...................................................................63 3.2.5 Effects of hypoxia-induced injury and treatment with GSNO on the phosphotyrosine proteome....................................................................................64 3.2.6 Bioinformatic analysis for network mapping........................................................65 3.2.7 GSNO inhibits cellular PTP activity via the S-nitrosylation.................................66 3.2.8 Application of PTP inhibitor mimics the effect of GSNO for protection of H9c2 cells against hypoxic injury....................................................................................67 3.2.9 Discussion.............................................................................................................68 3.2.10 Tables, schemes and figures................................................................................72 Chapter 4: Summary and Future Studies................................................84 4.1 Summary.......................................................................................................................85 4.1.1 The mechanism of S-nitrosylation of PTP........................................................85 4.1.2 The function of S-nitrosylation of PTP................................................................86 4.2 Future Studies...............................................................................................................86 Chapter 5: References...........................................................................................89 Appendix......................................................................................................................100 | |
dc.language.iso | en | |
dc.title | 酪氨酸磷酸水解酶亞硝基化作用機制及生理功能研究 | zh_TW |
dc.title | The Mechanism and Function of S-nitrosylation of Protein Tyrosine Phosphatases | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-1 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 邱繼輝 | |
dc.contributor.oralexamcommittee | 王寧,鄭敬楓,蔣輯武,陳光超 | |
dc.subject.keyword | 一氧化氮,亞硝基化修飾,酪氨酸磷酸水解酶,質譜分析,缺氧,冠狀動脈心臟病,酪氨酸磷酸化修飾,細胞骨架重組, | zh_TW |
dc.subject.keyword | Nitric oxide,S-nitrosylation,Protein tyrosine phosphatase,Mass analysis,Hypoxia,Coronary artery disease,Tyrosine phosphorylation,F-actin-organization, | en |
dc.relation.page | 100 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-02-11 | |
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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