酪氨酸磷酸化在誘發型一氧化氮合成酶蛋白質穩定度、二聚體形成、蛋白質交互作用中之角色探討

Role of tyrosine phosphorylation in mediating inducible nitric oxide synthase protein stability, dimer formation and protein-protein interaction

曾敬雅(Ching-Ya Tseng)

指導教授 ： 林庭慧

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00017

摘要

一氧化氮 (Nitric Oxide) 是一個與細胞訊息傳遞有關的氣體分子，由 L-arginine 經一氧化氮合成酶 (NOS) 催化而生成。當細胞面臨刺激時，誘發型一氧化氮合成酶 (iNOS) 會大量表現進而製造高濃度的一氧化氮，此高濃度的 NO 對細胞通常具有毒性。許多研究藉由抑制 iNOS 基因的轉錄或調控 iNOS 的後轉譯修飾，來降低 NO 對細胞的傷害。本論文主要探討酪氨酸磷酸化對 iNOS 蛋白質的穩定度、二聚體的形成以及蛋白質之間的交互作用中所扮演的角色。 我們發現 tyrosine kinase inhibitors 能有效的抑制脂多糖 (LPS) 和干擾素 (IFNγ) 所產生的 NO 含量、 iNOS 蛋白質表現量與降低 iNOS 蛋白質的穩定度，並且降低 iNOS 二聚體/單聚體比例。經由免疫沉澱的實驗發現在 MES-13 細胞內， iNOS 能與 caveolin 1 (cav 1) 和 heat shock protein 90 (hsp90) 產生交互作用；且此交互作用在加入 Genistein 、 PP1 或去除 iNOS 蛋白上酪氨酸的磷酸根，並不會受到影響。因此，在 MES-13 細胞內， tyrosine kinase inhibitors 可加速 iNOS 蛋白質的降解的機轉可能是透過減少其二聚體的形成有關，而並非依靠與 cav 1 之間的交互作用。

關鍵字

誘發型一氧化氮合成酶一氧化氮 ； 酪胺酸磷酸化 ； 蛋白穩定度 ； 二聚體形成 ； 蛋白質交互作用 ； 腎絲球細胞

並列摘要

Nitric oxide (NO), a messenger gas generated from L-arginine, is a product catalyzed by nitric oxide synthase (NOS). Inducible NOS (iNOS) produces high concentrations of NO when cells are challenged with endotoxins or cytokines. NO synthesis by iNOS was mostly regulated at transcriptional level and also by the post-translational modification of iNOS. It has also been reported recently that iNOS activity was mediated by tyrosine phosphorylation. In this present study, roles of tyrosine phosphorylation in mediating iNOS protein stability, dimer formation and protein-protein interactions were investigated in glomerular mesangial cell line, MES-13 cells. Tyrosine kinase inhibitors (either genistein or PP1) effectively inhibited lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced NO production and iNOS protein expression in MES-13 cells. In addition, both genistein and PP1 perturbed LPS/ IFNγ-induced iNOS protein stability in MES-13 cells. The dimer to monomer ratio of iNOS protein in MES-13 cells was attenuated with the addition of either genistein or PP1. Association of iNOS protein with caveolin 1 (cav 1) and heat shock protein 90 (hsp90) in MES-13 cells was confirmed by immunoprecipitation experiments. Neither genistein nor PP1 interrupted the interaction of iNOS protein with caveolin 1 in MES-13 cells. Furthermore, dephosphorylation of iNOS by LAR tyrosine phosphatase (LAR) did not affect its interaction with caveolin 1 in vitro. Our data indicated that tyrosine kinase inhibitors (either genistein or PP1) accelerated iNOS protein degradation through reducing dimer to monomer ratio, but not its interaction with caveolin 1 in MES-13 cells.

並列關鍵字

nitric oxide ； inducible nitric oxide synthase ； tyrosine phosphorylation ； protein stability ； dimer formation ； protein-protein interaction ； MES-13 cell

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