一個分選連接蛋白於TLR4調控的免疫反應上扮演的角色

The functional role of a sorting nexin family protein in TLR4-mediated immune response

趙俊豪(Chun Hao Chao)

指導教授 ： 徐立中

國立臺灣大學/醫學院/分子醫學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10757

摘要

先天免疫是宿主抵抗病原菌感染的第一道防線。TBK1為TLRs訊號傳遞下游調節干擾素產生的關鍵蛋白。在我們實驗室之前的研究中，利用酵母菌雙雜合系統篩選和TBK1有結合能力的蛋白，其中一個具結合能力的蛋白屬於分選連接蛋白家族。分選連接蛋白為細胞中囊泡運輸過程負責這種細胞膜形變的作用蛋白之一，然而，此類蛋白在TLR4的訊號傳遞上面所扮演的調控角色目前並不清楚，因此我們的研究主要想知道這個分選連接蛋白是怎麼去調節TLR4的訊號傳遞。首先，我們在293T細胞中利用免疫沉澱的實驗中確定了TBK1和此分選連接蛋白確實具有結合的能力，另外也可以在RAW 264.7細胞中利用螢光標定的方式看到兩者可以同時存在同一個區域。接著，我們在降低此分選連接蛋白的情況底下，觀察到在TLR4刺激後所調控的基因包括Ifn4a、Ifnb、Ccl5及Il6都減少了他們被轉譯的能力。另外，我們觀察到TLR4訊號傳遞最終兩個關鍵的轉路調控因子IRF3及p65入核的情況在此分選連接蛋白表現降低的情況下也受到了抑制。最後，我們檢測了幾個訊號傳遞過程中不同蛋白在此分選連接蛋白表現降低之後的活性，沒有太大的影響及變化。綜合以上的結果，我們認為此分選連接蛋白確實會調控TLR4的訊號傳遞過程，但詳細的分子機制我們還需要更多的努力來幫助我們解開這個問題。

關鍵字

先天免疫 ； 分選連接蛋白

並列摘要

Innate immunity is the first line of host defense against pathogen infection. TBK1 is a key player involved in TLRs-activated type I interferon production. Previous study in our lab used yeast two-hybrid screening and obtained a TBK1-interacted protein, a sorting nexin family protein. This protein family is a machinery protein involved in vesicle trafficking. However, the function of this sorting nexin protein in the regulation of TLR4 signaling is still unknown so we here aim to study the functional role of it in the regulation of TLR4 signaling. We first confirmed the association of the sorting nexin protein and TBK1 by co-immunoprecipitation assay in 293T cells. In addition, TBK1 colocalized with it in RAW 264.7 macrophages by immunofluorescent assay. Second, the expression of Ifn4a, Ifnb, Ccl5 and Il6 mRNA was downregulationed in the sorting nexin-depleted RAW 264.7 cells upon LPS stimulation. Third, the nuclear translocation of core transcription factors in TLR4 signaling, IRF3, was decreased in the sorting nexin-depleted cells after LPS stimulation. Furthermore, LPS-induced activation of MAPKs in the sorting nexin knockdown macrophages was not change. Taken together, our data suggest that this sorting nexin regulates TLR4-mediated immune response although the detailed mechanism still awaits further investigation.

並列關鍵字

innate immunity ； sorting nexin

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