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研究生:顧正崙
研究生(外文):Cheng-Lung Ku
論文名稱:人類乙型淋巴毒素受體訊息傳導之研究
論文名稱(外文):Study of Human Lymphotoxin beta Receptor Signaling
指導教授:謝世良
指導教授(外文):Shie-Liang Hsieh
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:82
中文關鍵詞:乙型淋巴毒素受體腫瘤壞死因子受體訊息傳遞
外文關鍵詞:Lymphotoxin beta receptorTNFRTRAFsignal transduction
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人類乙型淋巴毒素受體(LT-βR)是屬於腫瘤壞死因子受體群的成員,被發現會和腫瘤壞死因子成員LIGHT, LT-a1/b2和LT-a2/b1結合。LT-βR會影響周邊淋巴組織的發育及脾臟發生中心的生成;當LT-βR與配體結合或單株抗體刺激時,會引發與發炎有關的轉錄因子NF-kB活化和經由細胞凋零的方式引發細胞死亡。在LT-βR結構上,其細胞外的區域具有TNFR特有的半胱胺酸重複區域;但是其細胞內的區域則不具酵素活性或是與其他受體相似的區域,因此會與受體細胞內的區域結合的蛋白對於其訊息傳遞有很重要的功能。 TNF receptor associated factors (TRAFs)為一群會與TNFR受體群結合的蛋白,共有六個成員。其中TRAF2, 3和5會與LT-βR結合;其中TRAF3的dominant negative mutant會抑制LT-βR引發的細胞凋零,而TRAF5會參與其NF-kB活化。在本實驗室之前的研究中,LT-βR細胞內區域在氨基酸序列344 ~397具有自我結合的能力,並參與細胞自殘的訊息傳遞。此區域並會與兩個serine/ threonine 激 p50和p80結合並磷酸化LT-βR細胞內區域。為了進一步研究此區域自我結合能力與功能,我們利用過量表現的方式,發現過量表現LT-βR和其細胞內區域LT-βR(CD)都可以引發NF-kB活化,而這樣利用LT-βR(CD)引發之NF-kB活化可以被TRAF5 dominant negative mutant所抑制;這樣的現象說明LT-βR(CD)的自我結合能力足以引發受體活化NF-kB,而且其分子機制與受到配體所活化的機制相似。而利用deletion mutant,我們發現氨基酸序列344~397對於NF-kB活化是必要的;而利用yeast two hybrid系統,我們也發現此塊區域也會與TRAF5結合。之前的研究也指出此塊區域對於引發細胞凋零也是需要的,而我們亦發現參與細胞凋零訊息傳遞的TRAF3,也是結合在344~397這區域中。最後我們也利用點突變的方式研究受體的訊息傳遞,我們發現其中的一個突變EE mutant (E390A, E391A),會抑制其引發細胞凋零與加強NF-kB活化。而包括EE mutant與另外兩個突變PQS和PPP均會減低其對於 TRAF3的結合能力。綜合以上的實驗,我們發現LT-βR(CD)氨基酸序列344~397可引發NF-kB活化;與下游訊息分子TRAF3和5結合,而其中此區域的兩個氨基酸E390與E391對於其訊息傳遞與TRAF3的結合有重要的功能。因此我們相信此區域可能是個新的domain,並對於LT-βR訊息傳遞有非常重要的功能。
The human lymphotoxin beta receptor (LT-bR), a member of tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. It has been shown to be the receptor for LIGHT, LT-a1/b2 and LT-a2/b1. The signaling of LT-bR can induce NF-kB activation and /or cell death through apoptosis. However, the LT-βR intracellular domain is distinct from those of other TNFRs and lacks any inherent enzymatic activity. It is possible that the interaction with other proteins is important for LT-bR signaling. TNF receptor associated factor (TRAF) 2, 3 and 5 were associated with LT-bR and involved in LT-bR signaling. Previous data have shown that aa 344-397 of LT-βR (self-association domain) is important for LT-bR-induced apoptosis in overexpression system, and two proteins with serine/threonine kinase activity also associate with LT-bR(CD) aa 344-397 and phosphorylate LT-bR. In this study, we have observed that overexpression LT-bR or the cytoplasmic domain of LT-bR can induce NF-kB activation in 293 cells. The LT-bR(CD) induced NF-kB activation could be inhibited by TRAF5 dominant negative mutant. In deletion mutation analysis, LT-bR(CD) aa 344-397 is essential for ligand-independent LT-bR induced NF-kB activation, and the down stream signaling molecules TRAF3 and TRAF5 is also bind to LT-bR in the same region in yeast two hybrid system. To define the minimal region for the association of TRAF proteins, we use site-directed mutagenesis techinque to generated LT-βR(CD) mutants in this putative TRAF binding region. One of those LT-bR(CD) mutants, clone E390A and E391A, could reduce LT-bR (CD)-induced apoptosis in HeLa cell and enhance NF-kB activation, as well as abolish the binding affinity to TRAF3. Thus, LT-bR(CD) aa 344-397 is essential for its binding to TRAF3 and TRAF5, and plays a critical role in LT-βR signaling pathway.
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