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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  蛋白質體技術分析SARS冠狀病毒類木瓜蛋白酶影響第一型干擾素訊息途徑與誘導乙型轉化生長因子表現之研究

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/151746

標題: 蛋白質體技術分析SARS冠狀病毒類木瓜蛋白酶影響第一型干擾素訊息途徑與誘導乙型轉化生長因子表現之研究
Study on the Role of SARS-CoV Papain-Like Protease in Type I Interferon Signaling Pathway and Transforming Growth Factor-Beta1 Induction Using Proteomic Analysis
作者: 李詩雯
Li, Shih-wen
Contributors: 賴建成
Chien-Chen Lai
分子生物學研究所
關鍵字: 類木瓜蛋白酶干擾素;乙形轉化生長因子;蛋白質體
SARS;PLpro;interferon;TGF-beta;proteomics
日期: 2012
Issue Date: 2013-11-07 12:20:38 (UTC+8)
Publisher: 分子生物學研究所
摘要: 嚴重急性呼吸道症候群冠狀病毒(SARS Coronavirus,SARS-CoV)之類木瓜蛋白酶(PLpro)具有辨識LNGG序列之去泛素化(de-ubiquitining)的酵素活性,已被證實會藉由抑制干擾素調控因子3與NF-κB的活化,使抗病毒機轉受到抑制。本研究利用蛋白質體技術探討PLpro經第一型干擾素處理對人類前單核球細胞之訊息作用、細胞激素表現與蛋白質體變化之分析。PLpro可以抑制IFN調控的啟動子ISRE、AP-1與下游基因PKR、2’-5’-OAS、IL-6和IL-8活化,經二維電泳、質譜儀分析、西方墨點法與即時定量聚合酶連鎖反應驗證發現,PLpro可使ERK-1表現下調,並可透過促使ubiquitin-conjugating enzyme E2-25K及proteasome subunit alpha type 5的表現量上升而使得細胞ubiquitin-proteasome途徑活化。在蛋白酶體和ERK1/2抑制劑處理下,發現PLpro可增加ubiquitin-proteasome途徑以增加ERK1泛素化並降解,而IFN調控蛋白STAT1和cJun則受ERK1/2活化調控。此外,PLpro可以明顯的增加TGF-β1之mRNA與蛋白質產率。經二維電泳、質譜儀分析、西方墨點法與即時定量聚合酶連鎖反應驗證發現,PLpro可促使heat shock protein 27、protein disulfide-isomerase A3 precursor、vimentin、retinal dehydrogenase 2、glial fibrillary acidic protein、glutathione transferase Ω-1等TGF-β1相關蛋白與基因表現上調。在ERK1/2與蛋白酶抑制劑作用下,發現確實可以調控PLpro上調TGF-β1和vimentin的表現量。進一步,PLpro上調之HSP27與p38 MAPK及ERK1/2活化之訊息途徑有關聯,因此以p38 MAPK及ERK1/2抑制劑作用後可使PLpro誘導之TGF-β1、vimentin與第一型膠原蛋白(type I collagen)表現量下降。根據以上結果,證實了SARS-PLpro可透過上調ubiquitin-proteasome系統表現量與活化p38 MAPK及ERK1/2的訊息途徑進而促使TGF-β1表現,並藉著上調ubiquitin-proteasome系統表現量與下調ERK1表現量進而抑制由第一型干擾素活化之ERK1與STAT-1間的交互作用。
SARS coronavirus (SARS-CoV) papain-like protease (PLpro) recognizes a consensus motif LXGG as consensus cleavage sequence of cellular deubiquitinating enzymes and demonstrates inactivation of IRF3 and NF-κB, reduction of interferon (IFN) induction and suppression of type I IFN signaling pathway. This study investigates type I IFN antagonist mechanism, cytokine expression and proteomic change induced by PLpro in human promonocyte cells using proteomic analysis. PLpro significantly inhibited IFN-mediated promoter (ISRE, AP-1) and genes (PKR, 2’-5’-OAS, IL-6 and IL-8). 2-D electrophoresis, mass spectrometry, Western blotting and quantitative real-time PCR assays indicated PLpro decreased ERK1 expression and activated the ubiquitin proteasome pathway via up-regulation of ubiquitin-conjugating enzyme E2-25k and proteasome subunit alpha type 5. In addition, proteasome inhibitor and ERK1/2 inhibitor significantly reduced ERK1 expression and regulated STAT1 and c-Jun phosphorylation levels via activation of ERK1/2. Moreover, PLpro strongly increased the mRNA and protein expression of TGF-β1. 2DE/MS, Western blotting, ELISA and quantitative real-time PCR assays indicated PLpro up-regulating many TGF-β1-associated genes, including heat-shock protein 27, protein disulfide-isomerase A3 precursor, vimentin, retinal dehydrogenase 2, glial fibrillary acidic protein, and glutathione transferase Ω-1. ERK1/2 inhibitor and proteasome inhibitor significantly regulated the expression of TGF-β1 and vimentin in PLpro-expressing cells. Furthermore, PLpro up-regulated heat shock protein 27, linking with the activation of p38 MAPK and ERK1/2 signaling pathways. The treatment with p38 MAPK and ERK1/2 inhibitors significantly reduced PLpro-induced expression of TGF-β1, vimentin and type I collagen. Results demonstrated SARS PLpro promotes TGF-β1 expression through up-regulating ubiquitin proteasomal system and activation of p38 MAPK and ERK1/2 signaling pathway, and down-regulates ERK1 expression thus inhibiting the interaction between type I IFN activation of ERK1 and STAT1 via up-regulating of ubiquitin-proteasomal system.
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