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標題: | TIFA磷酸蘇氨酸與其FHA結構域分子間交互作用與活化NF-kappaB的機制 Intermolecular binding between TIFA-FHA & TIFA-pT9 mediates TNF-alpha stimulation & NF-kappaB activation |
作者: | Chia-Chi Huang 黃家琦 |
指導教授: | 蔡明道(Ming-Daw Tsai) |
關鍵字: | FHA結構域,轉錄因子NF-kappaB,磷酸化蘇氨酸,TIFA,細胞激素TNF-alpha傳遞訊號機制, FHA domain,NF-kappaB,phosphothreonine,TIFA,TNF-alpha-mediated signaling, |
出版年 : | 2012 |
學位: | 博士 |
摘要: | FHA (forkhead-associated) 是一種新發現的結構域,它可以專一性地結合磷酸化蘇氨酸,並藉此調控細胞內許多重要的機制。TIFA是所有已知人類蛋白質包含FHA結構域中最小的一個。先前的文獻已經發現在細胞中大量表現外源性TIFA會激活轉錄因子NF-kappaB,不過TIFA如何參與並調控這訊息傳導仍需要更深入的探討。在此研究中, 我們在TIFA上發現了一個蘇氨酸(第九個氨基酸)有被磷酸化修飾,並證實這個磷酸化蘇氨酸會與TIFA的FHA結構域結合。此交互作用會促進TIFA蛋白質間的聚合並激活下游的NF-kappaB。生化分析的結果顯示在緩衝溶液中,未磷酸化的TIFA是以二聚體的形式存在著,意味著TIFA FHA結構域和第九磷酸化蘇氨酸(pT9)間的結合是TIFA二聚體與二聚體間的交互作用。此外,我們也發現抑制內源性TIFA會削弱細胞激素TNF-alpha引發的訊息傳遞及其功能。根據這些結果,我們認為TIFA FHA-pT9的交互作用是TNF-alpha刺激後活化NF-kappaB傳導途徑中未知的新環節,而此TIFA二聚體分子間藉由FHA-pT9結合形成聚合體的機制,將是FHA結構域研究中另一個全新的範例。 The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with a FHA domain) is the smallest FHA-containing human protein. Its over-expression was previously suggested to provoke NF-kappaB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel phosphorylated threonine site, threonine 9 (pT9), on TIFA and show that this phosphorylation site binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-kappaB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer, and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of TNF-alpha-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-alpha stimulation and NF-kappaB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6603 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 生化科學研究所 |
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ntu-101-1.pdf | 9.76 MB | Adobe PDF | 檢視/開啟 |
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