IgE受器與PLCβ3蛋白-蛋白交互作用抑制劑之研究

Protein-protein interaction inhibitor between IgE receptor and PLCβ3

陳姿廷(Tzu-Ting Chen)

指導教授 ： 張學偉

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2014年)

摘要

過敏時IgE (Immunoglobulin E) 第一型接受器FcεRI (Fc epsilon RI) 扮演重要角色，最近有研究證明FcεRIβ會和下游蛋白PLCβ3 (phospholipase C beta 3) 交互作用，造成過敏物質的釋放。有趣的是，PLCβ3和FcεRIβ交互作用與細胞激素(cytokine) 產生相關，但是並不能抑制去顆粒化 (degranulation)。在慢性過敏症當中PLCβ3與FcεRIβ交互作用可提供一個治療標靶，因此找尋一個蛋白-蛋白交互作用抑制劑sil3589。以免疫沉澱法發現sil3589可有效地阻斷FcεRIβ與PLCβ3的交互作用，此外sil3589可抑制過敏發炎物質產生例如: Interleukin 4 (IL-4)、Interleukin 13 (IL-13) 、Cytokine-induced neutrophil chemoattractant 1 (CINC-1) 、Macrophage Inflammatory Protein 3 Alpha (MIP3α) 和 Tumor necrosis factor alpha (TNF-α)。sil3589也可增加負調控路徑 SHIP-1、 LYN蛋白質表現，以流式細胞儀和O-Phenylenediamine (OPD) 實驗，發現因阻斷了PLCβ3與FcεRIβ交互作用可能也干擾細胞膜上的動態平衡，更進一步研究內化 (internalization) 現象，當受到抗原刺激後sil3589可阻止FcεRI的內化。在動物實驗方面sil3589可有效減緩過敏原引發之氣喘反應，效果類似於類固醇，並減少各類發炎性免疫細胞肺臟浸潤。總結而論，本研究發現sil3589可阻斷FcεRIβ與PLCβ3交互作用，可抑制過敏發炎物質的釋放，因此sil3589可能做為有效的抗過敏性疾病藥物。

關鍵字

過敏 ； PLCβ3 ； 肥大細胞

並列摘要

The type I IgE receptor, FcεRI, is essential for hypersensitivity. Recent studies demonstrated that FcεRIβ interacts with its downstream factor, phospholipase C (PLC)-β3, mediating the allergic inflammatory responses. Interestingly, a function of FcεRIβ-PLCβ3 interaction is limited on inflammatory cytokines production but not on the immediate allergic degranulation. Therefore, the interaction between FcεRIβ and PLCβ3 may serve as an attractive therapeutic target for alleviating chronic inflammation. Here, the present study shows that a new protein-protein interaction (PPI) inhibitor, sil3589, is discovered and identified as a potent agent for treating allergic asthma. Using co-immunoprecipitation (Co-IP) assay, sil3589 effectively blocked FcεRIβ-PLCβ3 interaction, associated with inhibition of inflammatory cytokines such as Interleukin 4 (IL-4), Interleukin 13 (IL-13), Cytokine-induced neutrophil chemoattractant 1 (CINC-1), Macrophage inflammatory protein 3 alpha (MIP3α) and Tumor necrosis factor alpha (TNF-α). Consistently, the activity of negative regulatory effectors, Lyn and SHIP-1, was increased upon sil3589 treatment. Additionally, interruption of FcεRIβ-PLCβ3 interaction influenced the dynamics of FcεRI on cell surface, evidenced by flow cytometry analysis and O-Phenylenediamine (OPD) assay. sil3589 significantly inhibited antigen-stimulated FcεRI internalization, suggesting that Ag-IgE-FcεRI signaling cascade may be affected by sil3589. Lastly, in vivo experiment showed that sil3589 similarly to dexamethasone was able to alleviate antigen-induced asthmatic response. Furthermore, the influx of inflammatory cells was also reduced after receiving sil3589. Taken together, this study was the first to demonstrate that aiming at FcεRIβ-PLCβ3 interaction with sil3589 can therapeutically suppress allergic inflammation. Thus, sil3589 may be beneficial for allergic disease.

並列關鍵字

Allergy ； PLCβ3 ； mast cell

參考文獻

1. (1998), Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet ,1998,351, 1225-32.

2. Bateman, E.D., Hurd, S.S., Barnes, P.J., Bousquet, J., Drazen, J.M., FitzGerald, M., Gibson, P., Ohta, K., O'Byrne, P., Pedersen, S.E.,Pizzichini E., Sullivan, S.D.,Wenzel, S.E., Zar, H.J., Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J, 2008,31,143-178.

3. Berman, H., Henrick, K., Nakamura, H., and Markley, J.L., The worldwide Protein Data Bank (wwPDB): ensuring a single, uniform archive of PDB data. Nucleic Acids Res, 2007,35, D301-303.

4. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N., and Bourne, P.E. The Protein Data Bank. Nucleic Acids Res , 2000,28, 235-242.

5. Bernstein, D.I., Bernstein, C.K., Deng, C., Murphy, K.J., Bernstein, I.L., Bernstein, J.A., and Shukla, R., Evaluation of the clinical efficacy and safety of grapeseed extract in the treatment of fall seasonal allergic rhinitis: a pilot study. Ann Allergy Asthma Immunol ,2002,88, 272-278.