孟子青臺灣大學：生化科學研究所黃怡芬Huang, Yi-FenYi-FenHuang2007-11-262018-07-062007-11-262018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/52769一氧化氮(NO)透過 cGMP 路徑的方式來調控許多重要生理功能的研究至今相當成熟。然而，越來越多的報告顯示，NO可以不經由cGMP 路徑的方式來執行功能；其中最受到矚目的是對蛋白的半胱胺酸(cysteine)上進行後轉譯修飾，即半胱胺酸亞硝基化作用(S-nitrosylation)來調控。在眾多一氧化氮可能進行反應的目標中，酪胺酸去磷酸酶 (protein tyrosine phosphatases，簡稱PTPs) 由於其進行酵素催化作用的半胱胺酸(catalytic cysteine)具有特殊的低pKa，被認為很有機會進行半胱胺酸亞硝基化作用的反應。 本研究首先在體外(in vitro)系統中以PTP1B為對象(典型PTPs的代表，調控了許多重要的訊息路徑)，在分子層次上清楚說明了PTPs 亞硝基化作用發生的機制。結果顯示，利用一氧化氮給予者S-nitroso-N-acetylpenicillamine (SNAP)處理PTP1B造成了半胱胺酸可逆亞硝基化反應而抑制蛋白活性，但不會形成過度不可逆氧化而造成PTPs永久活性喪失的現象。另外，搭配質譜儀分析方法進一步驗證PTP1B Cys215的亞硝基化作用。此外，實驗證實在PTP1B 半胱胺酸亞硝基化作用的進行可以避免PTPs在H2O2的作用下所引起的蛋白過度氧化與永久失活，提供了在氧化爆起作用(oxidative burst)環境中一氧化氮對於PTPs保護功能的角色。本研究進一步針對了一氧化氮是否在細胞內廣泛地造成各種PTPs的亞硝基化作用進行深入的探討。主要是利用膠內酪胺酸去磷酸酶活性分析法(in-gel PTP activity assay)在EAhy926 內皮細胞中觀察內生性PTPs 氧化還原狀態。結果發現，不論是外源性一氧化氮(SNAP)或是在內生性一氧化氮 (VEGF)皆可以造成細胞內PTPs的亞硝基化作用，也發現麩胱甘肽(GSH)可有效還原亞硝基化作用的PTPs。結果也顯示，一氧化氮所引發的PTPs暫時性失去活性伴隨著細胞內蛋白酪胺酸磷酸化程度上升。綜合本研究的結果，不只顯示了PTPs 半胱胺酸亞硝基化作用重要的基本分子機制，並且證實了一氧化氮藉由亞硝基化作用影響PTPs活性而扮演重要細胞生理調控的角色。It has been well recognized that nitric oxide (NO) regulates important biological processes in a cGMP-dependent manner. In addition, substantial evidence has shown that NO may participate in the regulation of signal transduction through Cys S-nitrosylation of various signaling pathway. Among potential NO targets, the protein tyrosine phosphatases (PTPs) were proposed to be susceptible to nitorsylation due to the unique low pKa character of their essential catalytic Cys residue. In the present study, we initially explored the molecular mechanism for S-nitrosylation of PTP1B, a prototypic PTP which controls a diverse array of signaling pathways. Our results demonstrated that the treatment of PTP1B with S-nitroso-N-acetylpencillamine (SNAP), an NO donor, led to solely reversible inhibition, rather than facilitating the formation of permanently inactive form of phosphatase. In addition, the S-nitrosylated Cys residues of PTP1B were directly identified by advanced mass spectrometry analysis. Interestingly, S-nitrosylation of the catalytic Cys215 prevented PTP1B from irreversible oxidation, thus providing a protective effect for the phosphatase during an oxidative burst. We further investigated the regulation of PTPs by NO in a cellular context. For this purpose, an in-gel phosphatase activity assay was employed to analyze the redox status of endogeneous PTPs expressed in EAhy926 endothelial cells. We demonstrated that multiple PTPs were reversibly nitrosylated and inactivated in these ECs treated with either SNAP (exogenous NO donor) or VEGF (endogenous NO donor). Furthermore, cellular glutathione level played an essential role for an efficient reduction of nitrosylated PTPs. The data also show that the NO-mediated inactivation of PTPs was concomitant with an increased tyrosine phosphorylation level of cellular proteins. Our results not only reveal the fundamental basis for the mechanistic detail of Cys nitrosylation of PTPs, but offer insights into a novel biological role of NO that may govern tyrosine phosphorylation-dependent signaling through regulation of cellular PTPs.目錄 英文摘要……………………………………………………………………….1 中文摘要……………………………………………………………………….2 緒論…………………………………………………………………………….3 實驗材料……………………………………………………………………...13 實驗方法……………………………………………………………………...18 實驗結果……………………………………………………………………...25 討論……………………………………………………………………….…..38 圖表……………………………………………………………………….…..45 附圖…………………………………………………………………………...65 參考文獻……………………………………………………………………...722014420 bytesapplication/pdfen-US酪氨酸去磷酸酶一氧化氮Nitric oxideS-nitrosylationprotein tyrosineotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52769/1/ntu-95-R93b46012-1.pdf