Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21838
標題: 利用蛋白體學方法探討一氧化氮對細胞作用之影響與機轉
Exploring the Acting Mechanism of Nitric Oxide Using in-vitro Cell Cultures as a Model System by Proteomic Study
作者: 陳誼如
Chen, Yi-Ju
關鍵字: nitric oxide;一氧化氮;dinitrosyl-iron complex;quantitative proteomics;LC-MS/MS;protein S-nitrosylation;二亞硝基鐵錯合物;定量蛋白質體學;蛋白質半胱胺酸亞硝基化
出版社: 分子生物學研究所
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摘要: 
一氧化氮(NO)被認為是體內重要的訊息傳導分子,主要的功能包含血管舒張、神經訊息傳導、以及免疫反應等等。一氧化氮分子在體內極容易被分解而消失,因此通常被儲存在其他被稱為是一氧化氮攜帶者的分子內,例如S-亞硝基硫醇 (S-nitrosothiol) 、二亞硝基鐵錯合物 (dinitrosyl iron complex, DNIC)以及鐵硫亞硝基錯合物 (iron-sulfur cluster nitrosyl complex)。鐵硫亞硝基錯合物可細分為Roussins black salt (RBS)、Roussins red salt (RRS)及Roussins red ester (RRE)等,目前皆已經能夠以化學方法合成,並有相當多的生物方面的研究。然而,RRE鐵硫亞硝基錯合物因不具水溶性,使得吾人無法對其生物功能進行研究。近年來,清華大學化學所廖文峰老師實驗室已經成功合成出具極佳水溶性之RRE鐵硫亞硝基錯合物,提供對於RRE鐵硫亞硝基錯合物生物功能的極佳模式分子。為了解一氧化氮對生物作用的機制,在本研究中,吾人針對廖文峰老師實驗室合成的兩種水溶性鐵硫亞硝基錯合物(簡稱為DNIC-1及DNIC-2)與生化實驗中常用為一氧化氮提供者之標準品S-nitroso-N-acetyl- penicillamine (SNAP),進行一系列之生化功能測試及比較,並進一步針對其中一種水溶性鐵硫亞硝基錯合物DNIC-1進行定量蛋白質體分析。
於論文的第一部分,吾人選定一轉型之人類臍帶靜脈血管內皮細胞株EA.hy926為模式細胞,測試三種一氧化氮提供者分子之生物特性。此三種化合物並不具有顯著的細胞毒性(IC50>1 mM),同時皆能於細胞培養液中釋放一氧化氮分子。進一步分析發現DNIC-1分子與SNAP能與L-cysteine交互作用,促進一氧化氮的釋放,而DNIC-2分子則無此現象;此外DNIC-1與DNIC-2分子具有比SNAP更緩慢一氧化氮釋放能力,且釋放程度可隨劑量及作用時間升高。利用電子順磁共振(Electron Paramagnetic Resonance, EPR)偵測結果得知,DNIC-1可以藉由未知中間產物通過細胞。經由此三種化合物刺激後,細胞皆可被誘導產生熱休克蛋白(Heat shock protein 70, HSP70)的產生與蛋白質硝基化。綜合以上結果,吾人推定水溶性鐵硫亞硝基錯合物可作為一氧化氮提供者並調節細胞代謝,而DNIC-1也可作為蛋白質體研究之模式分子。
於論文的第二部分,吾人針對DNIC-1進一步地利用同位素親和性標定法(cleavable isotope-coded affinity tag,cICAT)之定量蛋白質體學研究方式,鑑定受到DNIC-1調控後表現量發生改變的蛋白質,並藉此探討一氧化氮所參與的作用機制。目前由定量蛋白質體學研究初步結果顯示,吾人成功辨識及定量695個蛋白質,其中13個蛋白質表現量增加,而17個蛋白質表現量下降。而這些表現量改變的蛋白質,與移除過氧化物及抗氧化效應等一氧化氮訊息傳遞因子有關,及可能與細胞吸附或移動有關。此外,EA.hy926細胞株中之蛋白質半胱胺酸亞硝基化與磷酸化表現量變化,與DNIC-1分子刺激細胞之時間點具高度相關性,並具有緊密的關聯性。綜合而言,本論文所發現之一氧化氮調控及可能參與途徑之作用機制,能使吾人更進一步了解內皮細胞中一氧化氮之生理角色與意義。

Nitric oxide, NO, has been recognized as an important biological message molecule, such as vasodilator, neurotransmitter, and immune assisting molecule. Because NO is extremely labile, it is usually stored in NO carriers such as S-nitrosothiols (RSNO), dinitrosyl-iron complex (DNIC) and iron-sulfur cluster nitrosyl complex. Several well-known iron-sulfur cluster nitrosyl complex, such as Roussin's black salt (RBS), Roussin's red salt (RRS) and Roussin's red ester (RRE), have been chemically synthesized in vitro. Among these complexes, RRE salts are usually insoluble in water therefore little is known about their biological functions. Recently, water-soluble iron-sulfur cluster nitrosyl complexes have been successfully synthesized by Dr. Wen-Feng Liaw's group in National Tsing-Hua University, and provided as model NO donors for biological studies in this study.
In this first part of thesis, biological effects of two water-soluble nitrosyl-iron complexes, designated as DNIC-1 and DNIC-2, were compared to a well-known NO donor, S-nitroso-N-acetyl- penicillamine (SNAP). A transformed human umbilical vein endothelial cell, EA.hy926, was chosen as a model biological system. Both DNIC-1 and DNIC-2 showed low cytotoxicity (IC50>1 mM), which is similar to SNAP. In vitro NO releasing assay showed that L-cysteine facilitated NO release from SNAP and DNIC-1, but not DNIC-2, in a dose and time-dependant manner. Electron Paramagnetic Resonance (EPR) analysis showed that EPR silent DNIC-1 can convert to an unknown EPR active form and transported into cells. In addition, both nitrosyl-iron complexes and SNAP induced cellular heat shock protein 70 (HSP70) expression, which have been shown previously to be mediated by NO treatment. In-vivo protein S-nitrosylation was also detected in all NO donor treated cells. Our data suggested that both DNIC-1 and DNIC-2 functioned as NO donors to regulate cellular metabolism. DNIC-1 may also serve a good model molecule to address how iron-sulfur-nitrosyl compounds regulate cellular metabolism.
To further study the proteomic change of endothelial cells in response to DNIC-1 treatment, in the second part of thesis, we took advantage of quantitative proteomics technology combining cICAT labeling and off-line multidimensional LC-MS. The preliminary quantitative proteomic results showed that 695 proteins were identified and quantified, among them 13 proteins were up-regulated and 17 proteins were down-regulated in DNIC-1 treated cells. Some of these regulated proteins have been found to be related to NO signaling such as super-oxide scavenging pathway and anti-oxidant effectors, as well as cell adhesion and motility. In addition, DNIC-1 also induced protein S-nitrosylation and phosphorylation in EA.hy926 cells time-dependent manner. Therefore, our data demonstrated a possible interplay of NO signaling and protein phosphorylation cascade. The acting mechanism as well as possible pathways regulated by NO deciphered from this study may allow us to better understand the physiological role of NO in endothelial cells.
URI: http://hdl.handle.net/11455/21838
其他識別: U0005-1508200715483100
Appears in Collections:分子生物學研究所

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